Microfluidic extrusion

ABSTRACT

A biopolymer fiber containing collagen. The biopolymer fiber has excellent ultimate tensile strength, modulus of elasticity, and strain at break comparable to native human tendons and ligaments. The fiber may substantially circular, ovoid, square, rectangular, ribbon-like, triangular, or irregularly shaped. The fiber exhibits an ordered, longitudinally-oriented structure, and the fiber allows infiltration of cellular growth. Implantable biopolymer scaffolds and sutures containing the fibers are provided as well as microfluidic and extrusion methods for producing the biopolymer fibers.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application claims the benefit of application Ser. No. 62/800,317,filed Feb. 1, 2019, the disclosure of which is hereby incorporated byreference in its entirety.

STATEMENT REGARDING GOVERNMENTAL SUPPORT

The data presented in this application was supported at least in part byDARPA Contract HR0011-15-9-0006. The US government has certain rights inthe invention.

BACKGROUND OF THE INVENTION 1. Field of the Disclosure

The present disclosure relates to a method for manufacturing collagenfibers and their incorporation into scaffolds and implantablebiocompatible devices prepared with such fibers. In particular, thedisclosure relates to a method for extruding collagen fibers havingsuperior mechanical strength, biocompatibility and immunologicalproperties.

2. Description of Related Art

Collagen is a fibrous insoluble protein consisting of bundles of tinyreticular fibrils. Collagen protein molecules combine to form white,glistening, inelastic fibers of the tendons, the ligaments, and thefascia. Collagen is found in connective tissue, including skin, bone,ligaments, and cartilage.

In particular, collagen fibrils combine to form tough connective tissuesuch as ligaments and tendons. Many efforts have been made tomanufacture collagen-containing tissue for use in the body to replacedamaged collagen body parts, including in particular ligaments andtendons. Such implantable devices may replace the damaged part directlyor may serve to provide a scaffold to facilitate repair of, andeventually replace, damaged soft tissues such as tendons and ligaments.

Such products must function in a variety of challenging biomechanicalenvironments in which multiple functional parameters must be addressed.These parameters include, for example, compatibility with bodily tissueand fluids, strength, flexibility, and biodegradability.

There is a need in the art for a system and method that addresses theshortcomings of the prior art discussed above.

SUMMARY OF THE INVENTION

In one aspect, the disclosure is directed to a biopolymer fibercomprising a collagen, wherein the biopolymer fiber has one or more ofthe following characteristics:

an ultimate tensile strength of between about 1 MPa to about 1,700 MPa;

a modulus of elasticity of between about 10 MPa to about 20,000 MPa;

a strain at break of between about 2 percent and about 45 percentelongation;

an average fiber diameter between about 10 μm and about 90 μm;

maintains its strength after soaking in DPBS at room temperature for atleast about 1 hour; and

wherein the filament exhibits an ordered, longitudinally orientedstructure.

In another aspect, the disclosure is directed to a bundle of thebiopolymer fibers comprising between 2 and about 10,000 fibers.

In still another aspect, the disclosure is directed to an implantablebiopolymer scaffold for supporting repair of a soft tissue injurycomprising the biopolymer fibers or the bundle.

The disclosure also is directed to a woven sheet-like support, a patch,or a brace comprising biopolymer fibers.

In yet another aspect, the disclosure is directed to a method forproducing a biopolymer fiber. The method comprises the steps of:

dissolving collagen in an acid solution to form a collagen solution;

passing the collagen solution at a first speed through a first needlehaving a first diameter simultaneously with passing a formation buffersolution at a second speed through a second needle coaxially surroundingthe first needle and having a second diameter greater than the firstdiameter to form a sheath around the collagen solution to form a coaxialflow,

wherein the second flow rate of the foundation buffer solution throughthe second needle is at least twice the first flow rate of the collagensolution through the first needle,

passing the coaxially-flowing collagen and formation buffer solutionthrough a reaction zone comprising a fibril-forming bath for a time andat speeds sufficient to form a fiber,

dehydrating the collagen fiber at an extrusion speed, and

withdrawing the fiber onto a spool at a third speed greater than theextrusion speed sufficient to increase molecular alignment and reducethe diameter of the fiber.

In another aspect, the disclosure is directed to a method for producinga biopolymer fiber. The method comprises the steps of:

dissolving collagen in an acid solution to form a collagen solution;

passing the collagen solution at a first speed through a first needlehaving a first diameter into a formation buffer solution,

passing the collagen and formation buffer solution through a reactionzone comprising a fibril-forming bath for a time and at speedssufficient to form a fiber,

dehydrating the collagen fiber at an extrusion speed, and

withdrawing the fiber onto a spool at a speed of between about 2 timesthe extrusion speed and about 10 times the extrusion speed sufficient toincrease molecular alignment and reduce the diameter of the fiber.

In yet another aspect, the disclosure is directed to a method forproducing a biopolymer fiber comprising the steps of:

dissolving clinical-grade collagen in an acid solution to form acollagen solution;

passing the collagen solution at a first volumetric flow rate through afirst needle to yield a first speed simultaneously with passing aformation buffer solution at a second speed in a tube coaxiallysurrounding the first needle and forming a sheath around the collagensolution to form a coaxial flow,

wherein the speed of the foundation buffer solution is between about 2times and about 20 times the first speed of the collagen solutionthrough the first needle,

passing the coaxially-flowing collagen and formation buffer solutionthrough a reaction zone comprising a fibril-forming bath for a time andat speeds sufficient to form a fiber,

dehydrating the collagen fiber at an extrusion speed, and

withdrawing the fiber at a third speed greater than the extrusion speedsufficient to increase molecular alignment and reduce the diameter ofthe fiber.

In a further aspect, the disclosure is directed to a method forproducing a biopolymer fiber comprising the steps of:

dissolving clinical-grade collagen in an acid solution to form acollagen solution;

extruding the solution through a nozzle into a guide that passes theextruded solution into a bath of formation buffer;

dehydrating fiber formed in the formation buffer bath; and

collecting the fiber.

In a still further aspect, the disclosure is directed to a method forproducing a biopolymer fiber comprising the steps of:

dissolving clinical-grade collagen in an acid solution to form acollagen solution;

passing the collagen solution at a first speed through a first needlehaving a first diameter into a formation buffer solution,

passing the collagen and formation buffer solution through a reactionzone comprising a fiber-forming bath for a time and at speeds sufficientto form a fiber,

dehydrating the collagen fiber at an extrusion speed, and

withdrawing the fiber onto a spool at a speed of between about 2 timesthe extrusion speed and about 12 times the extrusion speed, in one ormore stages, sufficient to increase molecular alignment and reduce thediameter of the fiber.

The disclosure also includes an aspect of providing an implantablebiopolymer scaffold for supporting repair of a soft tissue injurycomprising the biopolymer fibers, a method for supporting the repair ofa soft tissue injury comprising the implantation of the biopolymerscaffold.

Other systems, methods, features, and advantages of the invention willbe, or will become, apparent to one of ordinary skill in the art uponexamination of the following figures and detailed description. It isintended that all such additional systems, methods, features andadvantages be included within this description and this summary, bewithin the scope of the invention, and be protected by the followingclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention can be better understood with reference to the followingdrawings and description. The components in the figures are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention. Moreover, in the figures, likereference numerals designate corresponding parts throughout thedifferent views.

FIG. 1 is a schematic diagram of an embodiment of a method disclosed inthe specification;

FIG. 2 is a schematic diagram illustrating a first step in the formationof a collagen solution in an embodiment of the disclosure;

FIG. 3 is a schematic illustration of use of a degasserin collagenpreparation in an embodiment of a method disclosed in the specification;

FIG. 4 is a schematic illustration of a centrifuge suitable for use inan embodiment of a method disclosed herein;

FIG. 5 is a schematic illustration of centrifuge use in an embodiment ofthe disclosure;

FIG. 6 is a schematic illustration of an embodiment of coaxial needlesused to form collagen fiber in an embodiment;

FIG. 7 is a schematic illustration of a collagen fiber reaction zonecomprising a fibril-forming bath in an embodiment of the disclosure;

FIG. 8 is a schematic embodiment of a dehydration bath in an embodimentof the disclosure;

FIG. 9 is a schematic diagram of an embodiment of a device forseparating collagen fiber from a dehydrating bath;

FIG. 10 is a schematic diagram of a fiber collection system in anembodiment of the disclosure;

FIG. 11 is a schematic illustration of fiber collecting spools in anembodiment of the disclosure;

FIG. 12 is a schematic illustration of end treating in accordance withan embodiment of the disclosure;

FIG. 13 is a schematic illustration of a method of an embodiment of thedisclosure;

FIG. 14 is a schematic illustration of a method of an embodiment of thedisclosure;

FIG. 15 is a schematic illustration of an embodiment of an apparatussuitable for use to make product of the disclosure;

FIG. 16 illustrates additional details of the apparatus of FIG. 15;

FIG. 17 is a table summarizing compositions used in the disclosure;

FIG. 18A is the first of three sections of a table summarizingconditions used for embodiments;

FIG. 18B is the second of three sections of a table summarizingconditions used for embodiments;

FIG. 18C is the third of three sections of a table summarizingconditions used for embodiments;

FIG. 19 is a graph summarizing a mechanical property relevant to thedisclosure;

FIG. 20 is a graph summarizing another mechanical property relevant tothe disclosure;

FIG. 21 is a graph summarizing still another mechanical propertyrelevant to the disclosure;

FIG. 22 is magnified images of compositions manufactured in accordancewith the disclosure;

FIG. 23 is a graph summarizing the width of embodiments of thedisclosure;

FIG. 24 is a graph summarizing the thickness of embodiments of thedisclosure;

FIG. 25 is a graph summarizing a mechanical property of embodiments ofthe disclosure;

FIG. 26 is a graph summarizing another mechanical property ofembodiments of the disclosure;

FIG. 27 is a graph summarizing a relationship of a mechanical propertyof embodiments of the disclosure;

FIG. 28 is a graph summarizing a relationship of another mechanicalproperty of embodiments of the disclosure;

FIG. 29 is graphs summarizing properties of embodiments of thedisclosure;

FIG. 30 is images of embodiments of the disclosure;

FIG. 31 is a graph summarizing a property of embodiments of thedisclosure;

FIG. 32 is a graph summarizing another property of embodiments of thedisclosure;

FIG. 33 is a graph summarizing still another property of embodiments ofthe disclosure;

FIG. 34 is magnified images of an embodiment of the disclosure;

FIG. 35 is magnified images of a microfiber product;

FIG. 36 is magnified images of a control microfiber product;

FIG. 37 is an image of features of an embodiment of the disclosure;

FIG. 38 is an image of features of an embodiment of the disclosure;

FIG. 39 is a graph summarizing features of embodiments of thedisclosure;

FIG. 40 is a graph summarizing how size of an embodiment of thedisclosure changes with time;

FIG. 41 is a graph summarizing how a mechanical property of anembodiment of the disclosure changes with time;

FIG. 42 is a graph summarizing how another mechanical property of anembodiment of the disclosure changes with time;

FIG. 43 is a graph summarizing how yet another property of an embodimentof the disclosure changes with time;

FIG. 44 is a graph summarizing how still another property of anembodiment of the disclosure changes with time;

FIG. 45 is a table summarizing comparative information;

FIG. 46 illustrates an embodiment of a method of the disclosure;

FIG. 47 summarizes the properties and characteristics of an embodimentof the disclosure;

FIG. 48 summarizes the properties and characteristics of an embodimentof the disclosure;

FIG. 49 summarizes the properties and characteristics of an embodimentof the disclosure;

FIG. 50 summarizes the properties and characteristics of an embodimentof the disclosure;

FIG. 51 summarizes the properties and characteristics of an embodimentof the disclosure;

FIG. 52 summarizes the properties and characteristics of an embodimentof the disclosure;

FIG. 53 summarizes the properties and characteristics of an embodimentof the disclosure;

FIG. 54 summarizes the properties and characteristics of an embodimentof the disclosure;

FIG. 55 summarizes the properties and characteristics of an embodimentof the disclosure;

FIG. 56 summarizes the properties and characteristics of an embodimentof the disclosure;

FIG. 57 is an SEM image of an embodiments of the disclosure; and

FIG. 58 is an SEM image of an embodiments of the disclosure.

DETAILED DESCRIPTION

In one aspect, the disclosure is directed to a biopolymer fibercomprising collagen, wherein the biopolymer fiber has one or more of thefollowing characteristics:

an ultimate tensile strength of between about 1 MPa to about 1,700 MPa;

a modulus of elasticity of between about 10 MPa to about 20,000 MPa;

a strain at break of between about 2 percent and about 45 percentelongation;

an average fiber diameter between about 10 μm and about 90 μm;

maintains its strength after soaking in DPBS at room temperature for atleast about 1 hour; and

wherein the filament exhibits an ordered, longitudinally orientedstructure.

In another aspect, the disclosure is directed to an implantablebiopolymer scaffold for supporting repair of a soft tissue injurycomprising at least one biopolymer sheet comprising biopolymer fibers,wherein the biopolymer comprises collagen and the biopolymer fibers haveone or more of the following characteristics:

an ultimate tensile strength of between about 1 MPa to about 1,700 MPa;

a modulus of elasticity of between about 10 MPa to about 20,000 MPa;

a strain at break of between about 2 percent and about 45 percentelongation;

an average fiber diameter between about 10 μm and about 90 μm;

maintains its strength after soaking in DPBS at room temperature for atleast about 1 hour; and

wherein the filament exhibits an ordered, longitudinally orientedstructure.

The fibers exhibit an ordered, longitudinally-oriented structure, andallow cellular infiltration following implantation of the fibers, anddevices made with the inventive fibers, into a subject.

In another aspect, the disclosure includes an implantable biopolymerscaffold for repair or replacement of a human body part.

Biopolymer fiber typically is formed of collagen. In particular,telocollagen typically is obtained from any source (human, bovine,recombinants, jelly fish, etc.). Bio-acceptable polymer, such as silkfibroin; other types of collagen such as type II collagen;fibrin/fibrinogen; basement membrane proteins; hyaluronic acid, polyethylene oxide, poly ethylene glycol, poly caprolactone, polyethylene,polyhydroxybutyrate, PDLA; PDLLA and high molecular weight PDLLA; PLGA;and blends thereof, may be blended with collagen to form biopolymerfiber.

In still another aspect, the disclosure is directed to a method forproducing a biopolymer fiber comprising the steps of dissolving collagenin an acid solution to form a collagen solution. In one embodiment ofthis method, the collagen then is passed at a first speed through afirst needle having a first diameter to have a first speedsimultaneously with passing a formation buffer at a second volumetricflow rate through a second needle coaxially surrounding the first needleand having a second diameter greater than the first diameter to form asheath around the collagen solution to form a coaxial flow. The secondvolumetric flow rate of the formation buffer through the second needleis at least about twice the first volumetric flow rate of the collagensolution through the first needle.

The coaxially-flowing collagen and formation buffer are passed through areaction zone comprising a fibril-forming bath for a time and at speedssufficient to form a fiber, which is withdrawn onto a spool at a thirdspeed greater than the first speed, and typically twice the speed atwhich the fiber is extruded through the dehydration bath, sufficient toincrease molecular alignment and reduce the diameter of the fiber. Thefibers then may be cross-linked and dried.

In yet another aspect, the disclosure is directed to an alternativemethod for producing a biopolymer fiber. In this embodiment, a collagensolution is prepared and injected into a reaction zone in a comprising afiber-forming bath, such as a bath of formation buffer, for a time andat speeds sufficient to form a fiber. The fiber is withdrawn on a spoolat a speed between about 2 to about 10 times faster than the injectionspeed to increase molecular alignment and reduce the diameter of thefiber. The fibers then may be cross-linked and dried.

In various embodiments of the disclosure, collagen or collagen and othersuitable biopolymers are made into biopolymer or collagen fiber. Forease of understanding, the features of the disclosure will be describedas they relate to collagen. However, collagen may be blended or combinedwith suitable biopolymers in various combinations and proportions toobtain fibers of the type disclosed herein.

Throughout the specification, steps that might typically be takentogether during a typical manufacturing process, such as washing anddrying or soaking and drying, may be taken or repeated as appropriate toachieve a desired result. For example, in an embodiment, a compositionmay be washed and dried before advancing to the next step. In someembodiments, the material may be washed a second time and dried a secondtime before advancing to the next processing step, or may be washed asecond time, then advanced.

In other embodiments, a first washing or drying step may be madeoptional. Thus, a material typically washed, then dried, may go directlyto the drying step, and then moved on to the next processing step. Theskilled practitioner can recognize circumstances under which steps maybe repeated or eliminated.

The constructs, such as scaffolds, made from the fibers, allow cellularingrowth, that is, various types of cells from the animal into which thefiber (and devices made from the fiber) is implanted will grow into thepores of the scaffold, preferably aligned with the fibers in thescaffold. Constructs and scaffolds comprise single layer and multi-layerarticles that may be used as a substitute for a known repair feature,such as sutures used to re-attach body parts, for example opposing endsof a ruptured Achilles tendon. In addition to providing supportingstructures for use in repairing torn or damaged tendons, embodiments ofthe disclosure are suitable in ligament repair as well. Thus, otherexemplary ligaments for which the scaffolds or the present invention maybe used to provide support include the ACL, MCL, PCL, UCL, and otherhuman and animal ligaments. Other surgeries for which products of thedisclosure are useful include superior capsular reconstruction as atreatment option for superior rotator cuff tears, and in particular forotherwise irreparable or difficult to repair partial or full tears.Similarly, a multi-layered sheet may be used to overlap a repair tostrengthen it.

In particular, embodiments of the disclosure may be suitable for repairof ligaments, tendons, and other soft tissues of animals of all types.Collagen fibers of the disclosure may be used, for example, to reattachtorn ligaments and tendons, even those with only a partial tear. Pluralfibers also may be twisted, bundled, braided, interwoven, or otherwisearranged to improve a form factor that is easier to work with than asingle fiber is to manipulate, for example during surgery. Improving theform factor may make it easier to locate a fiber or platform accurately.Other form factors may be constructed to serve as a reinforcement orinternal brace for a torn natural body part. A brace connects from onebone to another bone to support a joint. Typically, a brace forms anisometric joint with restored biomechanics and the isometry of thenative joint.

The efficacy of collagen fiber produced in accord with embodiments ofthe disclosure may be illustrated by studying repairs made in, forexample, rabbits. In particular, reinforcements and internal brace andover-sewn structures of rabbit knees are suitable for evaluating theproperties and characteristics of collagen fiber of the disclosure andof structures made from this fiber.

FIG. 1 illustrates an embodiment of a system and method formanufacturing collagen fiber. The system and method may be described ascomprising four sections or manufacturing areas. A collagen solution isprepared in the first section, and collagen fiber is formed in thesecond section. The collagen fiber then is collected in the thirdsection and then may be post-processed to yield wet or dry collagenfiber in the fourth section, post-treatments or end of treatment.

The steps in the system and method illustrated in FIG. 1 may be groupedinto four categories, as follows:

Category Name Steps Included 1 Preparing Collagen Solution 105-120 2Forming Collagen Fiber 125-130 3 Collecting Collagen Fiber 135-150 4Post-Treatment or End Treatment 155-180

As seen at step 105 of FIG. 1, collagen is combined with an acidicsolution and stirred thoroughly at step 110. In some embodiments, theacid is between about 0.01 M and about 0.50 M acetic acid. In otherembodiments, the acid is between about 0.01 M and about 0.50 Mhydrochloric acid. The solution may be degassed at step 115, and thencentrifuged at step 120 to remove residual bubbles. Resultant collagensolution is extruded from a needle, and there may be a second needleco-axial therewith that supplies a formation buffer solution in step125. The resultant forming fiber may continue in through a formationtube in step 130. The resultant product is a formed collagen fiber.

The fiber then continues to a collection system, wherein the fiber isseparated from the formation buffer solution at step 135 and dehydratedat step 140. The collagen fiber is recovered at step 145 and air-driedat step 150. Then, post-processing may be carried out, as illustrated atstep 155, step 160, step 165, and step 170. Air-dried collagen fiber ona spool is submerged in cross-linking solution at step 155, optionallywashed at step 160, air-dried at step 165, and desiccated to form driedfiber at step 170. As illustrated in FIG. 1 by the dot-dash line,material may be optionally washed at step 160, dried at step 165, andreturned to wash step 160.

Alternatively, collagen is injected into a bath of formation solution toform a fiber. In this system, a second needle for coaxial injection offormation buffer is not necessary. Collagen thus injected is introducedto a collection system through dehydration at step 140. The fiber thenis processed in accordance with the remainder of the processing steps.

FIG. 1 provides a generalized view of a system and method for carryingout an embodiment of the disclosure. Additional details and disclosureare included in the following particular aspects and embodiments of thedescription.

In an embodiment, the disclosure is directed to a method for producing abiopolymer fiber comprising the steps of dissolving collagen in anacidic solution to form a collagen solution. The collagen then is passedat a first volumetric flow rate through a first needle having a firstdiameter to have a first speed simultaneously with passing a formationbuffer at a second volumetric flow rate through a second needlecoaxially surrounding the first needle and having a second diametergreater than the first diameter to form a sheath around the collagensolution to form a coaxial flow. The second volumetric flow rate of theformation buffer through the second needle is at least twice the firstvolumetric flow rate of the collagen solution through the first needle.

The coaxially-flowing collagen and formation buffer is passed through areaction zone comprising a fibril-forming bath for a time and atvolumetric flow rates sufficient to form a fiber, which is withdrawnonto a spool at a third speed greater than the first speed. The thirdspeed, typically about twice the speed at which the fiber is extrudedthrough the dehydration bath, is sufficient to increase molecularalignment and reduce the diameter of the fiber. The fibers then arecross-linked and dried.

In another embodiment, the disclosure is directed to an alternativemethod for producing a biopolymer fiber. Collagen solution is preparedand injected into a reaction zone in a comprising a fibril-forming bath,such as a bath of formation buffer, for a time and at speeds sufficientto form a fiber. The second needle to form coaxial flow of formationbuffer is not needed. Rather, collagen fiber is injected directly intothe fibril-forming bath, and then carried through the dehydration bath.

The fiber is carried through by being withdrawn on a spool at a speedbetween about 2 to about 4 times faster than the injection speed toincrease molecular alignment and reduce the diameter of the fiber. Thefibers then may be cross-linked and dried.

An embodiment of the method 1300 is summarized in FIG. 13. A collagensolution is prepared, as illustrated. A collagen solution is formed atstep 1305. A biopolymer may be mixed with the collagen. Collagen isdissolved in an acidic solution to form a viscous solution. The solutionis stirred at step 1310 to ensure thorough mixing. The mixed solutionmay have entrapped gas, and so may be degassed one or more times indegasser step 1315. The collagen solution then may be centrifuged, asillustrated at step 1320. Optionally, the degas/centrifuge steps may berepeated, as shown by the dot-dash lines on FIG. 1 and as feature 1316on FIG. 13, to reduce the volume of gas entrapped in the solution.

Thus-prepared collagen solution is formed into a collagen fiber bycoaxial extrusion with a formation buffer solution that serves as asheath for the fiber core, as shown at step 1325. The formation buffersolution volumetric flow rate typically is at least twice the volumetricflow rate of the forming collagen. This arrangement suppresses formationof individual fibrils; stretches and orients the fiber; and may smooththe surface of the fiber by imparting flow-induced crystallization tothe fiber.

The collagen fiber then is collected. As formation of the collagen fiberis completed at step 1330, the collagen then is separated from theformation buffer solution at step 1335 and dehydrated in a dehydratingsolution at step 1340. The dehydrated collagen then is collected on arotating spool in step 1345, which further stretches the fiber byrotating at a rate greater than, and typically about twice, the rate atwhich the fiber is supplied from dehydrating solution step 1340.Thus-collected fiber then is air-dried on the spool in step 1350.

In an alternative embodiment, collagen solution is formed into acollagen fiber by direct injection into formation buffer solution. Thus,step 1325 is skipped. The fiber is collected, separated from formationbuffer solution, and dehydrated in a dehydrating solution at step 1340.The fiber is collected on a rotating spool in step 1345, which collectsfiber at a speed of between about 2 times the formation speed and about4 times the formation speed.

Fiber that has been air-dried on the spool then may be post-processed.Fiber may be cross-linked in a cross-linking solution at step 1355, andthen may be rinsed at step 1360. The fiber then is air dried at step1365 and desiccated at step 1370 to yield dry cross-linked collagenfiber.

The equipment used in making collagen fiber is made of conventionalmaterials of construction suitable for resisting attack by any of theraw materials used to make collagen fiber in accordance with embodimentsof the disclosure. Metals, plastics, and other materials have propertiesand characteristics suitable to resist attack by raw materials,intermediates, solvents, and products during manufacture of collagenfiber.

Another aspect of the disclosure is directed to a collagen fiber havingone or more of the following characteristics:

an ultimate tensile strength of between about 1 MPa to about 1,700 MPa;

a modulus of elasticity of between about 10 MPa to about 20,000 MPa;

a strain at break of between about 4 percent and about 12 percentelongation;

an average fiber diameter between about 16 μm and about 70 μm; and

at least maintains its strength after soaking in biological fluid forabout 1 hour.

The fiber exhibits an ordered, longitudinally-oriented structure, andthe fiber allows infiltration of cellular growth.

A fiber of this embodiment is manufactured in accordance with the methodof an embodiment of the disclosure. Collagen may be obtained from manysources and in various forms. The quality of the collagen fiber may berelated to the quality of the raw material used. In some embodiments,bovine collagen typically is used. Bovine collagen may be obtained innatural form or as lyophilized powder.

Bovine collagen 202 may be made into a viscous solution 203 bydissolution in an acidic solution. Both mineral acids, such ashydrochloric acid, and organic acids, such as acetic acid, may be usedto prepare a collagen solution. For example, in an embodiment, Type Ibovine collagen with telopeptide ends intact may be dissolved in about0.01 M acetic acid to about 0.5 M acetic acid 201 in vessel 210 to forma viscous solution 203 comprising about 16 mg collagen/mL of solution.Solution concentrations may range from about 10 mg collagen/mL ofsolution to about 19 mg collagen/mL of solution. In another embodiment,lyophilized Type I bovine corium with telopeptide ends attached is mixedinto a mineral acid, such as HCl having a concentration of from about0.01 M to about 0.5 M, to form a solution having a concentration betweenabout 10 mg collagen/mL of solution to about 19 mg collagen/mL ofsolution, typically about 16 mg collagen/mL of solution.

In embodiments, collagen is allowed to dissolve for at least about 14hours, typically at least about 15 hours, and more typically at leastabout 16 hours. In some embodiments, collagen solution 301 is degassedin degasser 300 to remove bubbles from collagen solution 301. Screen 304ensures that collagen is not drawn out of the degasser through thedegasser gas flow exit 303. Degasser 303 typically is operated at apressure of between about 0 psia and about 3 psia. Collagen solution maybe exposed to up to about 2 degassing cycles, typically between about 1and about 2 cycles. Degassing removes gas bubbles that likely wouldinterfere with and disrupt extrusion of fibrous collagen.

Degassed collagen then may be further degassed in a centrifuge.Centrifuge 400 is illustrated with top 408 open, making bowl 403 visiblein FIG. 4. Tubes of material to be centrifuged and tubes used to ensurebalance of the centrifuge are placed into the wells in the rotating bowl409. Case 405 is sufficiently robust to contain any debris should any ofthe interior parts fail during use.

FIG. 5 illustrates centrifuge 500 having containment bowl 501 and lid508. The centrifuge rotates rapidly counterclockwise, as illustrated bymovement arrow 505. Tube 502 illustrates a tube before centrifugationcontaining collagen solution 503. As can be seen, the collagen solutionis homogeneous and has trapped bubbles in the otherwise homogenouscollagen solution 512.

Centrifugation at relative centrifugal force, or g values, between about400 rcf and about 4,000 rcf, typically between about 600 rcf and about1,000 rcf, and more typically between about 700 rcf and about 800 rcf,is suitable to reduce the entrapped bubble volume to essentially zerowithin between about 3 minutes and about 15 minutes, typically betweenabout 4 minutes and about 10 minutes, and more typically between about 5minutes and about 7 minutes.

In some embodiments, a pair of related steps may be repeated byalternating between the steps. For example, collagen may be processed indegasser 303 for one cycle, then in centrifuge 500 for 5 minutes, andthen returned to degasser 303 for a cycle, then centrifuged again for 5minutes. Operating in this alternative way may provide improvedefficiency. This improved efficiency may be realized by taking advantageof a shorter treatment time to achieve a given quantity of bubbles or toachieve a better result than linear processing may achieve.

Collagen then is coextruded with a solution to form collagen fiber.Extrusion of collagen solution at the core of a coaxial fluid may, insome embodiments, aid formation of a collagen fiber.

In some embodiments of the disclosure, collagen then is introduced tothe center of a coaxial flow needle, with a formation buffer solutionintroduced to the outer needle. Thus, the formation buffer solutionforms a sheath around the collagen. As illustrated in FIG. 6, collagensolution 650 is pumped through pump 655 and introduced into inner needle603 as collagen flow 601. Simultaneously, formation buffer solution 660is introduced to outer needle 604 as formation buffer solution flow 606.Outer needle 604 is coaxial with inner needle 603 so that formationbuffer solution forms a sheath around the central core of collagen. Asthe materials exit the needle to flow into a reaction zone comprising afibril-forming bath 701 (shown in FIG. 7), formation buffer solution 607has formed a sheath around collagen fiber 602, which begins to form as asolid fiber.

The diameter of a resultant product collagen fiber is made smaller thanthe inner diameter of the central needle by downstream processing. Thediameter of the central needle may be larger than the target diameter ofthe finished fiber. In some embodiments, the inner diameter of thecentral needle is between about 0.05 mm and about 100 mm; in someembodiments, the inner diameter of the central needle is between about0.1 mm and about 50 mm; in still other embodiments, the inner diameterof the central needle is between about 0.2 mm and about 20 mm; in yetother embodiments, the inner diameter of the central needle is betweenabout 0.3 mm and about 10 mm, and more typically between about 0.35 mmand about 5 mm. In some embodiments, an even narrower range of innerdiameter of the central needle, such as between about 0.03 mm and about10 mm, typically between about 0.10 mm and about 3 mm, still moretypically between about 0.30 mm and about 1 mm, and even more typicallybetween about 0.35 mm and about 0.50 mm.

In some embodiments, the inner diameter of the central needle is betweenabout 0.38 mm and about 0.44 mm, typically between about 0.39 mm andabout 0.43 mm, and more typically between about 0.40 mm and about 0.42mm.

In some embodiments, the inner diameter of the surrounding outer coaxialneedle that supplies formation buffer solution typically is betweenabout 1.95 times the inner diameter of the central needle and about 2.15times the inner diameter of the central needle, typically between about2.00 times the inner diameter of the central needle and about 2.10 timesthe inner diameter of the central needle, and more typically about 2.05times the inner diameter of the central needle.

In embodiments, formation buffer solution may be any solution that aidsformation of a collagen fiber. Formation buffer solution typically is asolution comprising TES, also known as2-[(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid orN-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid, together withsalts and buffering agents.

In some embodiments of the disclosure, formation buffer solution is WSB,a solution comprising 30 mM TES, 4.14 mg/mL sodium phosphate monobasicdihydrate, 12.1 mg/mL sodium phosphate dibasic heptahydrate, 135 mMNaCl, and 10 percent w/v PEG (polyethylene glycol). Similar solutionsalso may be suitable.

The flow rates of the collagen solution and of the formation buffersolution are adjusted so that the formation buffer solution sheathremains intact within the extrusion needles and reaction zone comprisinga fibril-forming bath. The speed of the formation buffer solution alsois established to be greater than the speed of the collagen solution soas to provide a stretch to the collagen fiber to improve the quality ofthe fiber. Indeed, in this way, the collagen will be urged to form arelatively straight, continuous fiber without kinks and other physicalshape aberrations. In some embodiments, fibers may be substantiallycircular, ovoid, square, rectangular, ribbon-like, triangular, orirregularly shaped.

In embodiments of the disclosure, the speed of formation buffer solutionin the needle reaction zone comprising a fiber-forming bath is higherthan the speed of the collagen solution. Formation buffer solution isused to neutralize the collagen solution and to assist withfibrillogenesis. Further, the higher speed of the formation buffersolution is used to pull or stretch the collagen stream, which createsan extensional field that helps align the collagen monomers in a processcalled flow-induced crystallization. This alignment helps collagenpolymerize and increases the strength of the resultant product.

In some embodiments, the volumetric flow rate of the formation buffersolution in the needle is between about 5 times the volumetric flow rateof the collagen solution in the needle and about 10 times the volumetricflow rate of the collagen solution in the needle, typically betweenabout 7 times the volumetric flow rate of the collagen solution in theneedle and about 9 times the volumetric flow rate of the collagensolution in the needle, and more typically between about 7.5 times thevolumetric flow rate of the collagen solution in the needle and about8.5 times the volumetric flow rate of the collagen solution in theneedle. In particular, 8 times the volumetric flow rate of the collagensolution in the needle is effective.

In embodiments, collagen stream 702 and formation buffer solution sheath707 enter reaction zone comprising a fibril-forming bath 701 of reactionsystem 700, as illustrated in FIG. 7. The reaction zone may have astructure, such as a formation tube, that forms reaction zone 701.However, typically, no structure need be present. The collagen fibercontinues to polymerize to form collagen fiber product as the streamsflow through reaction zone comprising a fibril-forming bath 701.Collagen fiber 752 and formation buffer solution 702 flow out ofreaction zone comprising a fibril-forming bath 701.

In embodiments, the speed of the collagen is adjusted to afford thecollagen a reaction or polymerization time of between about 15 secondsand about 60 seconds, typically between about 20 seconds and about 50seconds, and more typically between about 25 seconds and about 40seconds.

As shown in FIG. 8, in embodiments, at the end of the polymerizationperiod, collagen fiber 852 has formed and separates from formationbuffer solution 808 as the streams flow out of reaction zone comprisinga fibril-forming bath 701. The excess formation buffer solution flowsinto basin 801. Dehydration system 800 is so designed as to catchformation buffer solution 808 in basin 801 and to introduce collagenfiber 852 to dehydration bath 802.

Dehydration solution affords the opportunity to remove water from thecollagen fiber, reduce fiber diameter, and aid in fibrillogenesis. Inembodiments, dehydration solution comprises a solution of between about10 percent ethanol in MilliQ water and about 35 percent ethanol inMilliQ water, typically between about 15 percent ethanol in MilliQ waterand about 30 percent ethanol in MilliQ water, and more typically betweenabout 15 percent ethanol in MilliQ water and about 25 percent ethanol inMilliQ water. Skilled practitioners recognize that MilliQ water, alsowritten as Milli-Q water, is highly purified water produced in equipmentavailable from Millipore Sigma, Burlington, Mass. USA.

Collagen fiber 852 is passed through dehydration bath 802 for betweenabout 10 seconds and about 50 seconds, typically between about 15seconds and about 45 seconds, and more typically between about 20seconds and about 40 seconds. Throughout the period, collagen fiber 852remains submerged in dehydration bath 802. The volume of dehydrationbath 802 is between about 400 times the volume of formation buffersolution pumped per minute and about 800 times the volume of formationbuffer solution pumped per minute, typically between about 450 times thevolume of formation buffer solution pumped per minute and about 750times the volume of formation buffer solution pumped per minute, andmore typically between about 500 times the volume of formation buffersolution pumped per minute and about 700 times the volume of formationbuffer solution pumped per minute 601.

FIG. 9 illustrates the end part of the dehydration bath, from whichdehydrated collagen fiber is removed from the dehydration bath. As seenin embodiments illustrated in FIG. 9, dehydrated collagen fiber 930 isremoved from dehydration bath 802 at hook 910 on ring 920. As can beseen, hook 910 is retained in dehydration bath 802 by ring 920. Hook 910tends to aid in removal of dehydration bath from the dehydrated collagenfiber. Dehydrated collagen fiber 930 is pulled upwardly in the directionof arrow 940 by rotation of spool 1001, as shown on FIG. 10. Because thecollection speed of spool 1001 (FIG. 10) is greater than the extrusionflow rate of collagen fiber 930, hook 920, or a similar device, isappropriate to ensure that the collagen fiber remains submerged in thedehydration bath 802. As dehydrated collagen fiber 930 is lifted abovethe level of the dehydration bath, fluid droplets 905 can be seen to befalling off dehydrated collagen fiber 930.

FIG. 10 illustrates collection of dehydrated fiber onto spool 1001. Inembodiments, spool 1001 is rotated by motor 1011 in a clockwisedirection, as shown by arrow 1016. Spool 1001 is rotated at a speed thatprovides a draw ratio of between about 1.5 and about 3, typicallybetween about 1.75 and about 2.5, and more typically between about 1.90and 2.20. Similarly, spool 1002 is rotated at the same speed. The drawratio is the ratio between the spooling speed and the extrusion speed.Thus, there is a tension on first collagen fiber 1050 that pulls thefiber upward at hook 910. The fiber then is pulled above the wall ofdehydrating bath 802 and onto spool 1001.

Alternatively, in some embodiments of the disclosure, collagen isintroduced directly into a fibril-forming bath, without the coaxialneedles of FIG. 6 to form a coaxial flow illustrated in FIG. 7. Rather,collagen fiber is formed as collagen solution 852 is injected directlyfrom a needle into fiber-forming bath 870, after which processingproceeds as with the coaxial formation method which is an alternativeembodiment of the disclosure. The needle size for the collagen injectionis selected in the same way the needle size is selected for the coaxialinjection method, and the fiber is drawn through the fibril-formationbath and then into the dehydration bath in the same manner as otherembodiments. However, spool 1001 in FIG. 10 is rotated at a speed thatyields a draw speed of between about 2 times the fiber formation rateand about 4 times the fiber formation rate, typically between about 2.5times the fiber formation rate and about 3.5 times the fiber formationrate, and more typically between about 2.75 and 3.25 the fiber formationrate. Then, post-processing is carried out in the same manner as forother embodiments.

Arrow 1020 indicates passage of time for some embodiments, during whichspool 1001 has been translated relative to the position at the end ofdehydration bath 802 so as to form a single layer of fiber on the spool.Thus, spool rotation continues at the same speed and fiber 1052 is keptin tension as the spool is translated until spool 1002 is essentiallyfull. Time arrow 1030 illustrates a passage of time until the fibersupply is exhausted. Spool 1055 may then be recovered. The translationalspeed may be adjusted to adjust separation between fibers on a spool.

To ensure that tension is maintained on a fiber as the spool is rotated,typically the fiber is in contact with the entirety on the surface of aspool, such a spool 1110 used in some embodiments, as shown in FIG. 11.However, a spool need not have a continuous surface, as does spool 1110.In other embodiments, a number of rods could extend along the length ofthe spool. One such spool formed from rods is spool 1120 in FIG. 11,which comprises first rod 1125, second rod 1126, and third rod 1127. Therods provide sufficient surface to wind collagen thereon.

Fibers of the disclosure also may be chemically post-processed. FIG. 12illustrates potential post-processing steps. In embodiments, spool 1210containing collagen fiber is air-dried at 1220 for at least about 15minutes, typically at least about 20 minutes, and more typically atleast about 30 minutes. Air-dried fiber-containing tube 1210 then isplaced in a container for cross-linking. Typically, a container inembodiments of the disclosure minimizes the volume of the cross-linkingcontainer to reduce the amount of cross-linker required. Thus, asillustrated in FIG. 12, cylinder 1230 contains the amount ofcross-linking fluid necessary to cover cylindrical spool 1210, as shownat 1231. In embodiments, the volume of cross-linking solution per meterof fiber is at least about 3 μL, typically at least about 4.5 μL, andmore typically at least about 6 μL.

Fibers of the disclosure may be functionalized to provide amino groups,or, like collagen, may contain amino groups that can be crosslinked withaldehydes. Typically, small chain aldehydes, and more typically glyoxal(GLY) or with other conventional crosslinking reagents. For example,crosslinkers such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (EDC), N-hydroxysuccinimide (NHS), genipin,glyceraldehyde, glutaraldehyde, o-dextran, and low M procyanidin andhigh M procyanidin may be used. Alternatively, if the fiber isfunctionalized with carboxyl groups, then EDC and other carbodiimidesmay be used for crosslinking. Isocyanates react with both OH groups andamines. Therefore, isocyanate-based crosslinkers may be used tocrosslink the OH groups to each other within, for example, thefunctionalized PDLLA (linking an OH group to another OH group) toimprove media stability and strength. Isocyanates also may be used tolink collagen to OH groups in functionalized PDLLA via the NH₂ group(that is, amine group) from the collagen. Additionally,photocrosslinkers can be used.

The following reaction sequences are exemplary of cross-linkingreactions available in embodiments of the disclosure. In each of theexemplary reactions, P=polymer, which is the fiber in these reactions:

In particular, glyoxal provides suitable cross-linking in embodiments ofthe disclosure. In embodiments of the disclosure, a solution of 10 mMglyoxal in a solution of 70 percent ethanol and 30 percent MilliQ waterissued for cross-linking. The concentrations or proportions of thesecomponents may be varied to provide the desired cross-linking degree andfunctionality.

In embodiments of the disclosure, 0.25 mM EDC solution in the sheath maybe used as cross-linking solution.

As shown in FIG. 12, in some embodiments, tube and spool 1231 are rolledas illustrated schematically by arrow 1235. For example, the roller mayroll the tube and spool at about 1 RPM. Rolling continues for a timesufficient to obtain the desired degree of cross-linking. In someembodiments, at least about 24 hours is sufficient to obtain the desireddegree of cross-linking. Increasing cross-link time increases strengthof bonds within the fibers and improves stability of resultant product.Thus, in some embodiments, materials are allowed to cross-link for atleast about 48 hours, typically for at least about 72 hours.Cross-linking time of as much as about 1 month has been found toincrease cross-link strength even more. The container may be moved inany manner that ensures that the entire coil is submersed incross-linking fluid.

In some embodiments, the spool containing cross-linked collagen fiber1211 then is removed from the tube and optionally is placed in a MilliQwater rinse for about 10 min, as shown at rinse tank 1240 and arrow1221. Rinsed spool and fiber 1212 then are placed in a bath comprising100 mM glycine 1250 for a time sufficient to deactivate excess glyoxal.Typically, 10 minutes is sufficient. Removing glyoxal helps reducecytotoxicity of the fibers. Other cross-linking agents may be removed ina similar way, if necessary or appropriate.

In embodiments in which the rinsing step is skipped, spool and fiber1213 are placed in glycine at glycine bath 1250. Processing to dry fiberis as in embodiments with the rinse step.

Spool containing collagen fiber 1214 from glycine bath then is againrinsed in MilliQ water at tank 1260. In embodiments, 10 minutes issufficient to remove the glycine. Spool and fiber 1215 then is air-driedat 1270 for about an hour before being placed into a desiccating chamber1280 for about 24 hours. Dry, flexible fibers 1217 of FIG. 12 then arerecovered.

Embodiments of the disclosure are directed to a method 1300 in FIG. 13for producing a biopolymer fiber. In the embodiments, collagen isdissolved in an acid solution 1305 to form a collagen solution 1310. Insome embodiments, a compatible biopolymer is included with collagen. Thecollagen solution then may be degassed 1315, then centrifuged 1320 toobtain a collagen solution.

The collagen solution then is coextruded with formation buffer solutionas a sheath 1325. The collagen solution is passed at a first speedthrough a first needle having a first diameter simultaneously withpassing the formation buffer at a second speed through a second needlecoaxially surrounding the first needle and having a second diametergreater than the first diameter to form a sheath around the collagensolution to form a coaxial flow. The second speed of the foundationbuffer through the second needle is at least twice the first speed ofthe collagen solution through the first needle.

In some embodiments, the inner diameter of the central needle is betweenabout 0.05 mm and about 100 mm; in some embodiments, the inner diameterof the central needle is between about 0.1 mm and about 50 mm; in stillother embodiments, the inner diameter of the central needle is betweenabout 0.2 mm and about 20 mm; in yet other embodiments, the innerdiameter of the central needle is between about 0.3 mm and about 10 mm,and more typically between about 0.35 mm and about 5 mm. in someembodiments, an even narrower range of inner diameter of the centralneedle, such as between about 0.03 mm and about 10 mm, typically betweenabout 0.10 mm and about 3 mm, still more typically between about 0.30 mmand about 1 mm, and even more typically between about 0.35 mm and about0.50 mm.

In some embodiments, the inner diameter of the central needle is betweenabout 0.38 mm and about 0.44 mm, typically between about 0.39 mm andabout 0.43 mm, and more typically between about 0.40 mm and about 0.42mm.

In some embodiments, the inner diameter of the surrounding outer coaxialneedle that supplies formation buffer solution typically is betweenabout 1.95 times the inner diameter of the central needle and about 2.15times the inner diameter of the central needle, typically between about2.00 times the inner diameter of the central needle and about 2.10 timesthe inner diameter of the central needle, and more typically about 2.05times the inner diameter of the central needle.

In embodiments, formation buffer solution may be any solution that aidsformation of a collagen fiber. Formation buffer solution typically is asolution comprising TES, also known as2-[(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid orN-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid, together withsalts and buffering agents.

In some embodiments of the disclosure, formation buffer solution is WSB,a solution comprising 30 mM TES, 4.14 mg/mL sodium phosphate monobasicdihydrate, 12.1 mg/mL sodium phosphate dibasic heptahydrate, 135 mMNaCl, and 10 percent w/v PEG (polyethylene glycol). Similar solutionsalso may be suitable.

In some embodiments, the volumetric flow rate of the formation buffersolution in the needle is between about 5 times the volumetric flow rateof the collagen solution in the needle and about 10 times the volumetricflow rate of the collagen solution in the needle, typically betweenabout 7 times the volumetric flow rate of the collagen solution in theneedle and about 9 times the volumetric flow rate of the collagensolution in the needle, and more typically between about 7.5 times thevolumetric flow rate of the collagen solution in the needle and about8.5 times the volumetric flow rate of the collagen solution in theneedle. In particular, 8 times the volumetric flow rate of the collagensolution in the needle is effective.

In embodiments, the coaxially-flowing collagen and formation buffer flowthrough a reaction zone comprising a fibril-forming bath for a time andat speeds sufficient to form a fiber 1330. Formed collagen fiber then isseparated from the formation buffer solution 1335 and put into adehydrating solution 1340. Dehydration solution affords the opportunityto remove water from the collagen fiber, reduce fiber diameter, and aidin fibrillogenesis. In embodiments, dehydration solution comprises asolution of between about 10 percent ethanol in MilliQ water and about35 percent ethanol in MilliQ water, typically between about 15 percentethanol in MilliQ water and about 30 percent ethanol in MilliQ water,and more typically between about 15 percent ethanol in MilliQ water andabout 25 percent ethanol in MilliQ water.

In some embodiments, the fiber is withdrawn 1345 onto a spool at a thirdspeed greater than the first speed sufficient to increase molecularalignment and reduce the diameter of the fiber. This speed typically isat least about twice the speed at which the fiber flows through thedehydrating bath.

In other embodiments, step 1325 is skipped, and the coaxial sheathformation is not utilized. Rather, collagen solution is injected intoformation buffer solution, and motivated through the formation buffersolution and the dehydrating fluid by rotating the collection spools toprovide a rate that pulls the fiber at a speed between about 2 times theinjection speed and about 4 times the injection speed. The remainder ofthe steps, including potential post-processing, then are carried out.

In embodiments, the fibers are cross-linked in step 1355 after a shortair-drying period in step 1350. Typically, cross-linking is carried outin a glyoxal solution with agitation for a period sufficient to achievecross-linking. In embodiments, the fiber is left on the spool. It istypical to minimize the volume of the cross-linking container to reducethe amount of cross-linker required.

Cross-linking material may be any suitable cross-linker. In particular,glyoxal provides suitable cross-linking in embodiments of thedisclosure. In embodiments of the disclosure, a solution of 10 mMglyoxal in a solution of 70 percent ethanol and 30 percent MilliQ wateris used for cross-linking. The concentrations or proportions of thesecomponents may be varied to provide the desired cross-linking degree andfunctionality. In embodiments, the volume of cross-linking solution permeter of fiber is at least about 3 μL, typically at least about 4.5 μL,and more typically at least about 6 μL. A 24 hour cross-linking periodoften is suitable to achieve the amount of cross-linking. However,typically, a cross-linking period of at least about 48 hours providesincreased cross-linking, and a period of at least about 72 hoursprovides even more cross-linking.

The spool containing cross-linked collagen fiber then is removed fromthe cross-linking container and, in some embodiments of the disclosure,is placed in a MilliQ water rinse for about 10 minutes. In otherembodiments, the spool need not be rinsed. Spool and fiber then areplaced in a bath comprising 100 mM glycine bath step 1360 for a timesufficient to deactivate excess glyoxal. Typically, 10 minutes issufficient. Removing glyoxal may help reduce cytotoxicity of the fibers.

Other processing steps may be taken if glyoxal is not used as thecross-linking agent. The skilled practitioner will recognize appropriatepost-processing steps appropriate for these other cross-linking systems.

In embodiments, the spool containing collagen fiber from the glycinebath then is again rinsed in MilliQ water at step 1365. In embodiments,10 minutes is sufficient to remove the glycine. Spool and fiber 1214then is air-dried at step 1270 for about an hour before being placedinto a desiccating chamber 1370 for about 24 hours. Dry, flexible fibersare recovered.

In embodiments of the disclosure, the fiber produced is a biopolymerfiber comprising collagen. The biopolymer fiber has one or more of thefollowing characteristics:

an ultimate tensile strength of between about 20 MPa to about 170 MPa;

a modulus of elasticity of between about 200 MPa to about 3,500 MPa;

a strain at break of between about 4 percent and about 12 percentelongation;

an average fiber diameter between about 16 μm and about 70 μm afterdrying; and

at least maintains its strength after soaking in biological fluid forabout 1 hour.

The fiber exhibits an ordered, longitudinally-oriented structure, andthe fiber allows infiltration of cellular growth.

In another aspect, the disclosure is directed to an implantablebiopolymer scaffold for supporting repair of a soft tissue injury, orfor repair or replacement for a human body part. The scaffold comprisesat least one biopolymer sheet comprising biopolymer fibers, wherein thebiopolymer comprises collagen and the biopolymer fibers have one or moreof the following characteristics:

an ultimate tensile strength of between about 20 MPa to about 170 MPa;

a modulus of elasticity of between about 200 MPa to about 3,500 MPa;

a strain at break of between about 4 percent and about 12 percentelongation;

an average fiber diameter between about 16 μm and about 70 μm aftersoaking for about 1 hour in phosphate-buffered saline solution; and

at least maintains its strength after soaking in biological fluid forabout 24 hours.

The fiber exhibits an ordered, longitudinally-oriented structure, andallows infiltration of cellular growth. The sheet comprises fibersarranged in a typical way for convenience of handling during use. Forexample, a single fiber would be exceedingly difficult to use because ofthe small diameter. Thus, it is necessary or appropriate to formscaffolds, or structures larger than a single fiber, to providefiber-containing products suitable for repair or replacement of a bodypart. Thus, for example, it is possible to braid several fibers togetherto form a strand comprising collagen fibers. Such a strand may beuseful, for example, to oversew a rupture in a ligament or tendon. Theseand other uses will become apparent to the user.

Throughout the disclosure, testing of properties and characteristics iscarried out on 10 randomly-gathered fibers. Strength tests are carriedout with 10 fibers and a load of between about 0.3 N and about 2 N.

As noted herein, the stability of the collagen fiber is at leastmaintained, even after 1 hour in biologic solution. Further, additionalcross-linking achieved by continuing the cross-linking period to atleast about 48 hours, and even further to 72 hours, significantlyreduces swelling of the fiber and maintains or increases load capacity.

The following example is an example of an embodiment of the disclosureand is not meant to be limiting in any way.

Example 1

Collagen, type I bovine, with telopeptide ends intact, was removed frompackaging, and was combined with 0.05 M acetic acid to create a viscoussolution having a collagen concentration of 16 mg/mL. The solution wasallowed to dissolve collagen for 16 hours before being degassed forseveral cycles. Excess bubbles were removed by centrifuging at about 750rcf before and after degassing for 5 minutes. Collagen was aspiratedinto a 5 mL syringe and then attached to the center luer fitting of acoaxial needle (0.41 mm ID for collagen inlet and 0.84 mm ID forformation buffer inlet). The collagen syringe and coaxial needle werethen placed onto a syringe pump to be pumped at 60 μL/min.

The pH of formation buffer solution was adjusted to 8.0±0.1 and placedinto a covered beaker. The formation buffer solution was WSB, also knownas wet spinning buffer, a solution comprising 30 mM TES, 4.14 mg/mLsodium phosphate monobasic dihydrate, 12.1 mg/mL sodium phosphatedibasic heptahydrate, 135 mM NaCl, and 10 percent w/v PEG (polyethyleneglycol).

Tubing was placed at the bottom of the beaker and through a peristalticpump and then attached to the outer coaxial needle via a luer fitting,thus creating the outer sheath flow for the collagen. Formation bufferwas used to neutralize the collagen solution and to assist withfibrillogenesis. Formation buffer solution was flowed at 500 μL/min. Thefaster formation buffer was also used to pull or stretch the collagenstream, and created an extensional field that helps align the collagenmonomers in a process called flow-induced crystallization. Thisalignment helped collagen polymerize more readily and increased thestrength of the end product.

The collagen and formation buffer streams entered the reaction zonecomprising a fibril-forming bath, in which the fiber had time topolymerize and form a long chain. The reaction zone comprising afibril-forming bath ends at the inlet of a dehydration bath that caughtthe used formation buffer in a reservoir and allowed the fiber to travelroughly 45 cm through 20% ethanol and 80% MilliQ water. This bath helpedremove water from the collagen fiber, reduced its diameter, and aided infibrillogenesis. The bath was 2.5 cm wide and held roughly 300 mL ofsolution.

After the fiber traveled through the bath, it was then spooled onto a 50mm diameter spool that was 300 mm long and rotated at roughly 5 RPM,thus creating a draw ratio of approximately 2 (ratio between spoolingspeed and extrusion speed). This draw ratio helped further increase themolecular alignment and reduced fiber diameter which ultimatelyincreased strength. Translational speed of the spool was adjusted toalter the spacing between fibers.

The spools were allowed to air dry for at least 15 minutes before beingplaced into a cylindrical tube for crosslinking. The inner diameter ofthis tube was close to the outer diameter of the spool to reduce theamount of crosslinker required for full submersion. One-hundred twentymL of 10 mM glyoxal in 70% ethanol and 30% MilliQ water was prepared andpoured into the tube. The spool then was put into the tube. The tube andspool then were placed on a roller at approximately 1 RPM for 24 hours.

After 24 hours, the spool was removed from the tube and placed in aMilliQ bath for 10 minutes. The spool was then placed in a bath of 100mM glycine for 10 minutes to deactivate any excess glyoxal to helpreduce cytotoxicity, followed by a final bath of MilliQ water for 10minutes to remove any remaining glycine. The spool and fibers then wereair dried for approximately an hour before being placed into adesiccating chamber for 24 hours.

After desiccation, the fibers were dry and flexible which makes themeasily manipulated into useful shapes for building scaffolds. Resultingfibers had an average diameter of 25 μm and a tensile strength ofapproximately 100 MPa after a half hour soak in PBS. PBS, also known asphosphate-buffered saline, is a buffer solution commonly used inbiological research. It is a water-based salt solution containingdisodium hydrogen phosphate, sodium chloride, and, in some formulations,potassium chloride and potassium dihydrogen phosphate. The buffer helpsto maintain a constant pH. The osmolarity and ion concentrations of thesolutions match those of the human body (i.e., are isotonic).

Stability testing for 7 days in DMEM, a synthetic cell culture mediumcomprising amino acids, calcium chloride, potassium chloride, magnesiumsulfate, sodium chloride, and monosodium phosphate, glucose, andvitamins folic acid, nicotinamide, riboflavin, and B₁₂, at 37° C. showsa loss of approximately 25% original strength. DMEM also contains ironand phenol red for pH indication.

This example illustrates production of fiber within the scope of theclaims in accordance with a method within the scope of the claims. Thefiber will produce scaffolds, in accordance with the claims, for repairor replacement of human body parts.

Example 2

Collagen, type I bovine, with telopeptide ends intact, was removed frompackaging, and was combined with 10 mM hydrochloric acid to create aviscous solution having a collagen concentration of 16 mg/mL. Thesolution was allowed to dissolve collagen for 16 hours before beingcentrifuged at 733 rcf for 5 minutes. Excess bubbles are removed bydegassing for 2 minutes, and then centrifuging again at 733 rcf for 10minutes. Collagen was aspirated into a 20 mL syringe and then attachedto the center luer fitting of a coaxial needle (0.41 mm ID for collageninlet). The collagen needle was then placed onto a syringe pump to bepumped at 50 μL/min.

The pH of formation buffer solution was adjusted to 8.0±0.1 and placedinto a long bath. The formation buffer solution was WSB, also known aswet spinning buffer, a solution comprising 30 mM TES, 4.14 mg/mL sodiumphosphate monobasic dihydrate, 12.1 mg/mL sodium phosphate dibasicheptahydrate, 135 mM NaCl, and 10 percent w/v PEG (polyethylene glycol).

Formation buffer was used to neutralize the collagen solution and toassist with fibrillogenesis. Collagen was pumped into the formationbuffer solution and is guided through the bath. The collagen formationbuffer solution comprises the reaction zone, in which the fiber had timeto polymerize and form a long chain. The reaction zone comprising afibril-forming bath ends at the inlet of a dehydration bath of 20%ethanol and 80% MilliQ water through which the fiber is guided. Thisbath helped remove water from the collagen fiber, reduced its diameter,and aided in fibrillogenesis. Both baths were 2.5 cm wide and heldroughly 300 mL of solution.

After the fiber traveled through the baths, it was then spooled onto a50 mm diameter spool that was 300 mm long and rotated at roughly 10 RPM,thus creating a draw ratio of at least approximately 2 (ratio betweenspooling speed and extrusion speed). This draw ratio helped furtherincrease the molecular alignment and reduced fiber diameter whichultimately increased strength. Translational speed of the spool wasadjusted to alter the spacing between fibers.

The spools were allowed to air dry for at least 15 minutes but no morethan 1 hour before being placed into a cylindrical tube forcrosslinking. The inner diameter of this tube was close to the outerdiameter of the spool to reduce the amount of crosslinker required forfull submersion. One-hundred twenty mL of 10 mM Glyoxal in 70% ethanoland 30% MilliQ water was prepared and poured into the tube. The spoolthen was put into the tube. The tube and spool then were placed on aroller at approximately 1 RPM for at least 24 hours and up to 72 hours.

After 24 or up to 72 hours, the spool was removed from the tube and airdried for approximately an hour before being placed into a desiccatingchamber for 24 hours.

After desiccation, the fibers were dry and flexible which makes themeasily manipulated into useful shapes for building scaffolds. Resultingfibers had an average wet diameter of 30 μm and a tensile strength ofapproximately 120 MPa after a half hour soak in PBS. PBS, also known asphosphate-buffered saline, is a buffer solution commonly used inbiological research. It is a water-based salt solution containingdisodium hydrogen phosphate, sodium chloride, and, in some formulations,potassium chloride and potassium dihydrogen phosphate. The buffer helpsto maintain a constant pH. The osmolarity and ion concentrations of thesolutions match those of the human body (i.e., are isotonic).

Stability testing for 7 days in DMEM, a synthetic cell culture mediumcomprising amino acids, calcium chloride, potassium chloride, magnesiumsulfate, sodium chloride, and monosodium phosphate, glucose, andvitamins folic acid, nicotinamide, riboflavin, and B₁₂, at 37° C. showsa loss of approximately 25% original strength. DMEM also contains ironand phenol red for pH indication.

This example illustrates production of fiber within the scope of theclaims in accordance with a method within the scope of the claims. Thefiber will produce scaffolds, in accordance with the claims, for repairor replacement of human body parts.

Additional Disclosure and Comparative Information

In embodiments of the disclosure, clinical-grade atelocollagen andtelocollagen may be used to form microfluidics extruded collagenmicrofibers which then can be crosslinked with biological and benigncrosslinkers such as glyoxal or DL-Glyceraldehyde (DLG). Thesecross-linked fibers demonstrated hydrated ultimate tensile strength near300 MPa and modulus over 3 GPa, significantly stronger than 50 othercrosslinking strategies tested and exceeding native human Achillestendon and anterior cruciate ligament strength. Glyoxal cross-linkedfibers further retained 50% of the initial load-bearing capacity through3-6 months in culture. Collagen fibers implanted in rats demonstratedbiocompatibility, promoted the production of new, host-generated alignedcollagen growing along the fibers, and in the case of glyoxalcrosslinking, promoted an elevated pro-regenerative M2 macrophageresponse. Embodiments of the disclosure demonstrate marked improvementsin healing compared with other crosslinked fibers comprisingconventional and synthetic materials, making embodiments of thedisclosure superior fibers for generating strong collagen sutures or useas a device for ligament, tendon, or other soft tissue repairs.

Attempts to create materials suitable for tendon and ligament repairshave yet to produce a suitable product. To date, autografts, allografts,and synthetic materials as sutures, braces, or grafts for soft tissueclosure or joining, for example, have been found to have significantclinical limitations. Allografts such as dead, decellularized, andchemically treated implants, can be slow to integrate, inflammatory, andmay possibly delay healing (Seon, Song and Park, 2006). Synthetic graftscan break down into acidic byproducts damaging surrounding tissue(Taylor et al., 1994; van Sliedregt et al., 1994; Matsusue et al.,1995). Synthetic grafts often do not match the mechanical or materialproperties of tendons or ligaments (Hogan et al., 2015), which may leadto generation of stress risers and creation of a debilitatingnon-isometry if used in a joint space. Autografting extends surgery timeand associated trauma (e.g. blood loss, risk of infection) due to theneed for a second procedure to recover the autologous tissue, causingadditional trauma in the process (Chen et al., 2009; Perrone et al.,2017). Joint reconstruction with autografting or allografting furtherresults in a higher incidence and severity of premature osteoarthritis,thus affecting the quality of life (Leiter et al., 2014; Smith et al.,2014; Perrone et al., 2017). Rising rates of post-traumaticosteoarthritis has become a significant problem for military veterans(Showery et al., 2016).

There remains an unmet need in manufacturing an ideal biological,strong, material for tendon and ligament repair sutures and forresorbable sutures. Synthetic non-resorbable suture withcollagen-coating (e.g. Collagen-Coated FiberWire®) has been madeavailable in an attempt to improve biocompatibility, reduce inflammationand reduce abrasiveness from the strong synthetic materials,particularly for orthopedic indications.

Crosslinked fibers extruded from type I collagen may produce strongproduct. However, these products are unsatisfactory and presentbiological, strength, and other objections. For example, mostcrosslinkers are cytotoxic, use harsh chemicals foreign to the body, andare also not used in currently marked U.S. Food and Drug Administration(FDA) approved or cleared products, making their use more challengingfor clinical translation. In addition to their potential use inaugmenting ACL or AT repair, braided collagen fibers have the potentialto be used as sutures for general, ocular, and plastic and cosmeticsurgery if shown to have high uniform tensile properties, consistentuniform diameters, biocompatibility and controllable resorption withregenerative capacity.

Embodiments of the disclosure are directed to a novelmicrofluidic-extrusion system to produce microfibers of clinical type Icollagen as filaments and as thin ribbon-like structures. Embodiments ofthe disclosure satisfy rigorous mechanical, biochemical,cytocompatibility, and biocompatibility criteria, making fiberembodiments of the disclosure having properties specifically forbiomedical use. Embodiments of the disclosure exhibiting order from themolecular-scale through mesoscale and up to macroscales required toproduce useful products, these collagen fibers disclosed herein havepotential applications in tendon and ligament repair, wound closure, andother indications where an advanced collagen-suture-based biomaterialmay be beneficial across the fields of surgery in medicine.

FIG. 14 illustrates schematically manufacture of collagen microfiber inaccordance with embodiments of the disclosure, and potential biomedicalapplications of suitable products. Freeze-dried collagen is dissolved inacid in step 1401, wherein collagen molecules 1402 are obtained.Extruded microfibers 1403 are twisted in spinneret 1404 to form twistedmicrofibers 1405. The microfibers comprise assembled molecular collagen1406. The collagen may be spooled at step 1407.

Collagen may be more suitably used in three-dimensional structuresformed by twisting or braiding individual fibers. Braided fibers 1411 ortwisted fibers 1405 then may be used to suture tear 1415 in an anteriorcruciate ligament (ACL) in knee 1412 of a patient. Collagen ACL sutures1414 are used to repair the tear, and collagen skin sutures 1415 may beused to close the wound.

Any number of fibers may be associated, whether twisted or not, to forma bundle, and bundles may be assembled into larger bundles. For example,bundles may comprise between 2 fibers and about 10,000 fibers, orbetween about 4 fibers and about 6,000 fibers, typically between about 8fibers and about 4,000 fibers, and more typically between about 12fibers and about 2,000 fibers. Then, bundles may be combined, bytwisting or otherwise, to form larger bundles. Bundles that are combinedneed not have equal numbers of fibers.

Bundles may be described by the number of fibers in the bundle. Forexample, a 5-fiber bundle may be called a penta-fiber; 8 fibers wouldproduce an octa-fiber, and so on. Systems and equipment with othernumbers of nozzles or extruders may be used to produce such bundles.

FIG. 15 and FIG. 16 illustrate a method for obtaining collagen fiber anda system in which the reactions can be carried out. In embodiments ofthe disclosure, up to 2% (w/v) clinical grade lyophilized telocollagen(Telo) or atelocollagen (Atelo) (Collagen Solutions, CA) ormethacrylated collagen (Advanced BioMatrix, CA) was dissolved in up to0.05 M acid (most typically acetic or hydrochloric) overnight byagitation. As shown in system 1500, acidified collagen 1501 was thenpumped through the center of a nozzle system. The system may includecoaxially arranged conduits or needles 1503. Neutralizing alkalineformation phosphate buffer containing salts (Sodium chloride, SodiumPhosphate Dibasic, Sodium Phosphate Monobasic, andN-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid) and PEG(polyethylene glycol) was pumped 1501 through the system in the outerportion of the coaxial conduit 1503. The formation buffer ran at avolumetric flow rate that is between about 5 times and about 20 times,typically between about 8 times and 15 times, and most typically aboutten times the rate at which collagen was introduced, which causes theprotein to be extended and partially aligned, imparting mechanicalstrength to the resulting fiber 1504. The fiber became more solid as itpassed through the formation tube before entering a bath 1505 of 20%aqueous ethanol. In addition to dehydrating the fiber, this bath helpedremove residual formation buffer thus contributing to improved strengthand stability of the resultant collagen microfiber. After dehydration1506, the microfiber 1507 may collected on two-bar device 1508. Othersuitable collections devices also can be used.

In some embodiments, acidified collagen fibers may be formed byextrusion before entering the formation bath. For example, acidifiedcollagen fibers may be formed by use of spinneret 1404. In someembodiments, a plurality of syringes of acidified collagen may be formedsimultaneously.

FIG. 46 illustrates system 4600, which uses an array of syringes to formacidified collagen fibers. System 4600 may be considered to be ahigh-throughput system. In some embodiments of the disclosure, clinicalgrade collagen is dissolved in acid (acetic acid or a mineral acid suchas HCl) in a closed container made of materials inert to the acid and tothe collagen. Polypropylene is one such material. The volume of thecollagen and acid typically is less than about 50% of the volume of theclosed container to encourage thorough mixing. The solution is stirredovernight, or for between about 16 hours and 30 hours, typically betweenabout 15 hours and about 20 hours. The solution then is centrifuged todegas the solution.

The degassed solution then is placed into syringes. The number ofsyringes used equals the number of fibers to be formed simultaneously.System 4600 in FIG. 46 shows use of outlets from 8 syringes mounted inrotatable plate 4601. The plunger of each syringe is pressed into thebarrel of the syringe by a plate (not shown) to ensure that the fibersare extruded in essentially equal quantities. Acidified collagen ispressed through first nozzle 4602 to form first fiber 4612; throughsecond nozzle 4603 to form second fiber 4613; through third nozzle 4604to form third fiber 4614; through fourth nozzle 4605 to form fourthfiber 4615; through fifth nozzle 4606 to form fifth fiber 4616; andthrough the remaining nozzles. In some embodiments, not all nozzles areused. In embodiments of the disclosure, rotatable plate 4601 may havemore or fewer nozzles mounted through it.

The fibers are gathered at guide 4630 and fed into formation buffer bath4640. The fibers are kept taught after extrusion. In some embodiments,rotatable plate 4601 may be turned in either direction to producetwisted fiber. In system 4600, rotatable plate 4601 may be rotated byrotation of drive plate 4620, which meshes with rosette notches 4621.Any suitable drive system may be used. In some embodiments, rotatableplate 4601 is not rotated, so the resultant bundle of fibers is nottwisted. However, the bundle is maintained under tension by a tensioneron the fiber bundle as the fibers are dehydrated, and until it is woundon a collector. Typically, a grooved cylinder is a suitable collector,particularly for wet fibers.

FIG. 16 illustrates details of the nozzle system 1600. Pump 1502 pumpsliquid acidified collagen 1620 into the center needle of biaxial needle1503. Buffer solution, also called sheathing solution, from 1610 isintroduced in the direction of flow arrow 1611 into the outer needle ofbiaxial needle 1503, thus forming collagen microfibers 1504 as thepolymerization process proceeds. The collagen fluid is focused by thesheathing fluid 1611 in extensional flow. The detailed view of theneedle illustrates how acidified liquid collagen 1620 moves in thedirection of the low speed arrows, and then increases in speed, asindicated by high speed arrows 1630 and higher speed arrows 1640.Similarly, sheathing fluid moved in the direction of speed arrows 1611,arrows 1635, and arrows 1645. Flow of the reactants continues in thedirection of arrow 1650, where the shading indicates how buffer(sheathing) fluid 1670, which contains phosphate, interacts with thecollagen solution and removes water 1680 from the collagen stream.

Once the microfibers 1507 were collected onto device 1508, they wereair-dried for half hour and subsequently crosslinked under differentexperimental conditions. Chemical reagents used during extrusion andcrosslinking are included in Table 1 in FIG. 17.

In situ crosslinking (chemical or enzymatic) for the groups shown inTable 2 in FIG. 18 was performed by dissolving the amount shown in FIG.18 for each crosslinker in acidified collagen mixture for time stated inFIG. 18. Concentrations and times of crosslinking for some materialswere obtained from specific references identified in FIG. 18. FIG. 18summarizes strength comparisons. Conditions in italics were selected forcharacterization post optimization of collection methods. Microfibersfrom in situ crosslinked collagen were then extruded on to a two-bardevice and kept taut as shown in FIG. 15. FIG. 18 also shows that thecross-linker can be present in an amount between about 5 mM and about500 mM, typically between about 10 mM and about 500 mM, and moretypically between about 25 mM and about 250 mM.

More typically, however, un-crosslinked microfibers may be collected onsolid spool 1110 (see FIG. 11) with closely spaced grooves. Microfiberswere collected directly onto these grooves while maintaining tautness.Collection onto spools typically is more efficient than the two-bardevice. Spools of un-cross-linked microfibers were cross-linkedchemically in 70% aqueous ethanol as used for the 2-bar device. Tubecontaining the microfiber spool in crosslinker solution was placed onrollers, as shown in FIG. 12, and rotated at 1 rpm to ensure uniformcrosslinking of microfibers.

For post-extrusion chemical crosslinking, un-crosslinked or in situcrosslinked and taut collagen microfibers that were extruded on two-bardevice 1508 or grooved roller 1110 were air dried for half hour and thensubmerged into a solution of crosslinker in 70% ethanol solution andplaced on a rocker at low speed. The aqueous ethanol medium ensured thatmicrofibers remained dehydrated throughout the crosslinking period.After crosslinking, microfibers were stored in a desiccator untilfurther tests were performed.

In some embodiments, collagen fiber is wet or damp when collected. Insuch cases, the fibers may tend to stick to each other if they areallowed to touch, especially during collection. Thus, in someembodiments, a two- or multi-bar collector device may be advantageouslyused because it may allow the fibers to dry before being contacted byanother fiber. In some embodiments, a grooved roller is particularlyuseful for collection of wet fibers because fiber-to-fiber contact isprecluded, as only 1 fiber is collected in a groove.

In some embodiments, the fiber may be dried by blowing a gas, typicallyair, over the fiber after it leaves the dehydration bath and before thefiber is collected. The fiber is suspended between the dehydration bathand a collector, which may be a flat cylinder, a bobbin, or any othersuitable collector. The fibers need not be kept apart from each other asthey are dry.

In some embodiments, the fiber may be dried by passing air at roomtemperature over the fiber at room temperature and at a speedbag 0.25m/sec and about 10 m/sec, typically between about 1 m/sec and about 4m/sec, and more typically about 2 m/sec. The speed of the drying airshould not be so high as to rupture, tear, or break, the fiber. The airis passed over for about the time it takes to dry the fiber, typicallyequal to the time it takes the fiber to travel about 1 meter. The dryingair may be moved by fans in an open system or in a recycling system. Insome embodiments, the collection device, such as a bobbin or a flatcylinder. The cylinder rotates at a draw speed between about 1 time andabout 9 times the formation speed. In such a circumstance, essentiallyinfinitely long fibers may be made.

The dehydrothermal treatment (DHT) for crosslinking microfibers involveddehydrating relaxed extruded microfibers at 110° C. and under vacuum for1, 3, and 5 days with or without additional crosslinking in glyoxal, asdescribed above.

For UltraViolet Radiation (UVR) mediated crosslinking, methacrylatedcollagen was used for extrusion. The extruded microfibers were thenexposed to a 365 nm emitting UV light source for 20 minutes. Thesemicrofibers were then placed in a desiccator or further crosslinked with10 mM glyoxal in 70% aqueous ethanol.

Mechanical properties of single microfibers were generated using a“discrete fiber” test method wherein the cross-sectional area ofindividual microfibers and a known quantity of microfibers on acartridge are averaged to determine the ultimate tensile strength (UTS),modulus, and strain at failure (%), because a single microfiber was toodelicate to consistently handle. While two-bar collection setup 1508 ledto microfibers being cylindrical, the microfibers collected on thesolid, grooved spool were thin and ribbon-like. The ribbon-like collagenfibers have a width between about 10 μm and about 70 μm, typicallybetween about 15 μm and about 60 μm, and more typically between about 20μm and about 50 μm. The ribbon-like collagen fibers have a thicknessbetween about 4 μm and 20 μm, typically between about 5 μm and about 18μm, and most typically between about 6 μm and about 17 μm.

Widths were measured from analyzing images obtained at 10 differentpoints on 3 separate, 1.5-inch long, microfibers using an inverted lightmicroscope, such as Axio Vert.A1 Model, Zeiss, Germany, and ImageJsoftware (NIH Shareware, Bethesda, Md.). Cross-sectional images ofmicrofiber bundles using a Scanning Electron Microscope (SEM) were usedto determine the thickness of the microfiber using Image J software. Inorder to meet the demands of rigorous mechanical testing that would berelevant with regard to the performance of embodiments of collagenmicrofibers of the disclosure in vivo, a high-throughput method ofwet-tensile-testing our microfiber samples, such as that disclosed inGentleman et al., 2003, may be used.

A bath and sample holding system was used to provide wet-tensilestrength mechanical testing of bundles in a cartridge. This system madeit possible to provide a 30-minute soak while processing a sample every5 minutes during soaking of extruded microfiber embodiments of thedisclosure. The soaking fluid may be Gibco's Dulbecco'sphosphate-buffered saline (DPBS), available from ThermoFisherScientific. Typically, a minimum of 4 cartridges were mechanically wettested at room temperature under uniaxial tensile testing on an MTSCriterion Model 42 (Eden Prairie, Minn.) at a pulling rate of 1 mm/s toobtain stress vs strain curves. Discrete-fiber testing was done togenerate results quickly while optimizing processing parameters.

The bath and sample holding system includes a bath filled sufficientlyto cover the materials being tested in fluid. The fluid may be Gibco'sDulbecco's phosphate-buffered saline (DPBS). During testing, the sampleholder was held in fluid by jaws at opposite ends of the tensile tester.Testing was carried out by moving the jaws away from each other.

The UTS of wet embodiments of the disclosure typically is between about1 MPa and about 800 MPa, typically between about 75 MPa and about 400MPa, and more typically between about 90 MPa and about 350 MPa, and evenmore typically between about 100 MPa and about 325 MPa. The modulus ofwet embodiments of the disclosure is between about 10 MPa and about7,500 MPa, typically between about 100 MPa and about 6,000 MPa, and moretypically between about 1,000 and 4,000 MPa.

The UTS of dry embodiments of the disclosure typically is between about25 MPa and about 1,900 MPa, typically between about 100 MPa and about1,800 MPa, and more typically between about 5000 MPa and about 1,700MPa, and even more typically between about 1,200 MPa and about 1,700MPa. The modulus of dry embodiments of the disclosure is between about14,000 MPa and about 20,000 MPa, typically between about 15,000 MPa andabout 19,000 MPa, and more typically between about 15,500 and 18,500MPa.

Testing to compare certain embodiments of wet and dry fibers showedcomparative ranges of Ultimate Tensile Strength of about 1 to about 755MPa for wet fibers vs. about 25 to about 1650 MPa for dry fibers; aModulus of Elasticity of about 10 to 7,200 MPa for wet fibers vs. about15,950 to about 18,600 MPa for dry fibers; a Strain at Break of about 2to about 41% for dry fibers vs. about 9 to about 14% for dry fibers; andan Average Fiber Diameter of about 14 to about 82 μm for wet fibers vs.about 10 to about 70 μm for dry fibers.

SEM imaging was used to obtain cross-sectional as well as longitudinalmicrostructural signatures of un-cross-linked and cross-linked extrudedmicrofibers. SEM imaging was performed using a Zeiss Evo 10 microscope(Zeiss) with a 10 kV beam intensity. For cross-sections, microfiberbundles were soaked in DPBS for 30 minutes, dried for an hour on SEMstubs, sputter coated, and imaged.

For TEM, dry microfibers from Telo GLY group (telocollagen cross-linkedwith glyoxal) were re-hydrated using distilled water. These were thenfixed in 2% glutaraldehyde (Electron Microscopy Sciences, PA) and 4%paraformaldehyde (Alfa Aesar, MA) at room temperature for 30 minutes.Subsequently, 2 washes (10 minutes for each wash) using cacodylatebuffer (Electron Microscopy Sciences) was done. This was followed by a30-minute incubation in 1% Osmium Tetroxide (Electron MicroscopySciences), one wash in cacodylate buffer and 2 washes in distilled water(10 minutes each). Dehydration through a series of ascending ethanolconcentrations (once in 30%, 50%, 70% and 95% for 10 minutes and twicein 100% for 10 minutes each wash) was performed; microfibers were thenimmersed twice in 1:1 mixture of ethanol and propylene oxide (ElectronMicroscopy Sciences) mixture for 10 minutes, followed by 100% propyleneoxide treatment for 10 minutes. These samples were left overnight in 1:1EPON 812:propylene oxide (Electron Microscopy Sciences). EPON 812 is aglycerol-based aliphatic epoxy resin. Next day, the samples wereimmersed in 4:1 EPON 812:propylene oxide for 4 hours and transferred to100% EPON 812 for overnight incubation. Next day, samples weretransferred to fresh EPON 812 resin, embedded into bullet capsules(Electron Microscopy Sciences) and polymerized at 60° C. for 12 hours.The molds were thinly sectioned and imaged using TEM (Model No. Jeol1230, Jeol USA, MA). Alternative methods for determining these valuesmay be used.

Ninhydrin assay may be used to evaluate the amount of free amino groupsin cross-linked microfibers. For this, un-cross-linked as well ascrosslinked microfibers were cut into lengths ranging from 14-16 cmeach. At the same time, various known concentrations of a standard aminoacid, glycine (Sigma-Aldrich), were prepared in 0.05% acetic acidaccording to manufacturer's protocol. The microfiber samples and glycinesolutions were heated in ninhydrin solution (Sigma-Aldrich) for 20minutes followed by cooling to room temperature for at least 1.5 hours.Then, 95% ethanol was added to each of the samples and glycinestandards. Optical absorbance of these samples was recorded with anultraviolet-visible spectrophotometer (SpectraMax i3, Molecular Devices,ODU, Norfolk, Va.) at 570 nm. Other methods of testing may be used.

Absorbance of various known glycine concentrations was used to obtain astandard curve. The amount of free amino groups in un-crosslinkedsamples (M_(UX)) and crosslinked (M_(X)) microfibers is proportional tothe optical absorbance of the solution and was obtained from thestandard curve of glycine that was generated. In order to calculateDegree of crosslinking, Equation 1 was used, as follows:

$\begin{matrix}{{{Crosslinking}\mspace{14mu}{Degree}\mspace{14mu}(\%)} = {\begin{pmatrix}{M_{UX} - M_{X}} \\M_{UX}\end{pmatrix} \times 100}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

Differential Scanning calorimetry (DSC) and Fourier-Transform Infrared(FTIR) Spectroscopy were used to determine whether amide bondscharacteristic of type I collagen were present. Testing of microfiberswas performed using a Differential Scanning calorimeter (DSC2500, TAInstruments, DE) and FTIR spectroscopy was performed on Platinum ATR(Brucker, Billercia, MA). FTIR spectra was used to confirm the presenceof three major peaks of amide bonds characteristic of type I collagen at1235 cm⁻¹, 1560 cm⁻¹, and 1650 cm⁻¹ wavelengths. Un-cross-linked andcross-linked microfibers were compared to the starting material byassessing shifts in peaks with the Essential FTIR bioinformaticssoftware (Operant, Madison, Wis.).

Extruded microfluidic fibers as single fibers, bundles of 150microfibers (held together by coated Vicryl 4-0 (Ethicon, NJ) suture andcut to a final size of 10 mm), or on cartridges used for mechanicaltesting as described above were sealed inside Tyvek pouches with aSTERRAD chemical indicator (4MD Medical Solutions, Lakewood, N.J.) andsent for E-beam sterilization (Steri-Tek, Fremont, Calif.) using a 20KGy+/−2 KGy target dose.

Sterilized glyoxal and DL-Glyceraldehyde crosslinked microfibers werehydrated in tenocyte growth media for 30 minutes and placed in 24 wellplates that were pre-coated with Poly(2-hydroxyethyl methacrylate)(pHEMA) (Sigma Aldrich). Twenty-five thousand human tenocytes (ZenBio,NC) (in 100 μl tenocyte growth media) were seeded on sterilizedmicrofibers in triplicates. After seeding, cells were allowed to attachfor 1 hour before an additional 500 μl of tenocyte growth media wasadded. After 12 days, tenocyte attached microfibers were stained withlive cellular stain, CellTracker™ Green CMFDA (5-chloromethylfluoresceindiacetate) (Thermo Fisher Scientific) following manufacturer's protocol.Samples were then fixed using 4% paraformaldehyde and subsequentlystained with a nuclear stain, DAPI (Thermo Fisher Scientific) tovisualize attached tenocytes on microfibers using a confocal microscope(Zeiss Axio Observer Z1, Zeiss).

Cytotoxicity (or cell viability) of embodiments of extruded microfiberson human tenocytes was assessed using the CyQuant Lactate Dehydrogenase(LDH) cytotoxicity assay kit (Invitrogen) and MTT assay kit (SigmaAldrich) following manufacturer's protocol. Briefly, after determiningthe optimum seeding density for the assay, 7×10³ tenocytes were platedon each well of 48 well plates and allowed to grow for 24 hours intenocyte growth media in a humidified incubator maintained at 37° C. and5% CO₂. Sterilized microfiber bundles were rinsed for 10 minutes in cellculture media and placed on tenocytes in each well. Tenocytes grown onplastic (cells only) were used as positive (for cell survival orviability). Zinc dibutyldithiocarbamate (ZDBC) film and 10 mM glyoxalchemical were used as negative (for cell survival or viability)controls. The effects of Ethicon Vicryl suture were also assessed inthis experiment as it was used to hold extruded microfiber bundlestogether. Wells seeded with tenocytes but containing no samples were setup to evaluate the maximum and spontaneous LDH release as described inthe manufacturer's protocol. Samples were incubated for 7 days beforeevaluating the release of LDH in the media. The % cytotoxicity using LDHassay was calculated following manufacturer's protocol. The % CellSurvival was then calculated as 100-% Cytotoxicity. In embodiments ofthe disclosure, % cell survivability is at least about 94%, typically atleast about 95%, more typically at least about 96%, and most typicallyat least about 97%. It is also possible to achieve 98% or 99% cellsurvivability. The % Cell viability using MTT assay was calculatedfollowing manufacturer's protocol. In embodiments of the disclosure, the% Cell viability is at least about 70%, typically at least about 80%,more typically at least about 85%, and most typically at least about90%. Other suitable test methods are available

The health and viability of live tenocytes growing with extrudedmicrofiber embodiments of the disclosure was also assessed using theAlamarBlue™ assay (Bio-Rad, Hercules, Calif.) as per manufacturer'sprotocol.

Embodiments of cross-linked microfiber bundles were subcutaneouslyimplanted into rats. All surgical procedures were conducted according toa protocol approved by Institutional Animal Care and Use Committee(IACUC), Old Dominion University, Norfolk, Va. Per ISO 10993-6, n=3crosslinked collagen microfiber bundles (prepared and sterilized asdescribed above) or collagen coated FiberWire® (suture control) wereimplanted subcutaneously in female Sprague Dawley rats. Rats wereanesthetized with isoflurane inhalation. Flanks were shaved, and Nairdepilatory cream was applied to remove hair from surgical site.Incisions were made dorsally in the flank area and a hemostat was usedto create a pocket for implants. Once scaffolds were placed in thepocket, the incision was closed using suture. After 4 weeks, the ratswere humanely euthanized for tissue collection.

Harvested microfiber explants at 4 weeks were fixed in 4%paraformaldehyde (Alfa Aesar) for 24 hours then transferred to DPBS(Thermo Fisher Scientific). The samples were sectioned to obtain 5 μmthickness and serial sections were stained with hematoxylin & eosin(H&E) as well as Masson's Trichrome at IDEXX (West Sacramento).Polarized light microscopy was used to image collagen organization inthe tissues surrounding the implants.

Immunolabeling was also performed on serial sections to detect thepresence of CCR7 (M1) and CD163 (M2) macrophage phenotypes in nativetissues surrounding our implants using standard protocols provided byantibody manufacturers. Briefly, after deparaffinization, antigenretrieval (20 minutes boiling in 10 mM Citrate Buffer pH 6),permeabilization and blocking with 2.5% horse serum, slides were stainedfor either CD163 (M2 macrophage phenotype), or CCR7 (M1 macrophagephenotype). The M2 macrophage marker, mouse anti-rat CD163 (#MCA342GA,BioRad, CA), was diluted to 1:30 for an overnight incubation inhumidified chamber. Post incubation, slides were washed in PBS andincubated with a goat anti-mouse secondary antibody (#A-11005, ThermoFisher Scientific) at a 1:50 dilution for 1-hour in the dark at roomtemperature. CCR7, a M1 macrophage marker, was diluted at 1:50 in PBSfor an overnight incubation (#MA5-31992, Thermo Fisher Scientific). Nextday, after PBS wash steps (3 times), a goat anti-rabbit fluorescentantibody (#A32740, Thermo Fisher Scientific) was applied to the slidesat a concentration of 1:200 for 1-hour in the dark at room temperature.For primary controls, serum-blocked slides were either stained with IgGMouse (1:30) (Thermo Fisher Scientific) and goat anti-mouse secondaryantibody (1:50) or IgG Rabbit (1:200) (Thermo Fisher Scientific) andgoat anti-rabbit secondary antibody (1:200). For secondary controls,serum-blocked slides were stained with only the secondary fluorescentantibodies. All antibodies were diluted in blocking serum. All slideswere stained for the nucleus with DAPI for 5 minutes, washed in PBS andmounted using VectaMount (Vector Labs, CA) for visualization andanalysis.

The immunolabeled slides were examined and imaged using an invertedlight microscope (Axio Vert.A1 Model, Zeiss). Fluorescence images wereacquired for the test and control slides (data not shown) under sameexposure conditions. The images for the test samples were evaluated.Quantitative analysis was performed to obtain the number of cellsexpressing M1 only, M2 only, M1 and M2, and/or no M1/M2 phenotype. Here4-5 areas per image (3 images were analyzed per test sample) ofapproximately 20-30 μm at the interface of the implants and nativetissue (2-3 cell layers) were analyzed using a high-power microscopefield (40× magnification). The total number of cells was determined bycounting DAPI stained nuclei. The number of cells labeled positively foreach marker(s) was also counted. The proportion of cells that werelabeled with the specific marker(s) was determined as a percentage oftotal number of cells in that region.

Embodiments of the disclosure also were subjected to long term stabilitytesting. Telo GLY microfibers were de-spooled under tension ontocartridges. Six sterilized cartridges were hydrated and mechanicallytested as described above to obtain mechanical properties of themicrofibers prior to incubating the remainder of the sterilizedcartridges in a petri dish containing Eagle's Minimum Essential Medium(EMEM) (ATCC, VA) supplemented with 1% Gibco® Antibiotic-Antimycotic(ABAM) (Thermo Fisher Scientific) to suppress bacterial and fungalcontamination in an incubator maintained at 37° C. and 5% CO₂.Throughout the duration of the experiment, it was ensured that thecartridges were always submerged in sterile contamination-free media andhence remain hydrated. Six soaked cartridges were removed at 1 week, 1month, 3 months, and 6 months to perform MTS testing. Simultaneously,microfiber diameters were measured (as described above) to determine theextent of swelling of the microfibers overtime.

An unpaired two tail t-test was used to assess any significantdifferences in a property or characteristic between any two groups. Atwo-way ANOVA followed by the post-hoc Tukey's Multiple Comparison Testalso were used to assess differences in UTS for different crosslinkergroups in Table 1 in FIG. 17. Ordinary one-way ANOVA followed byDunnett's multiple comparisons test also was performed to assessdifferences in health and viability, as described in additional detailbelow. A priori, p values <0.05 were defined as significant. All testswere performed using GraphPad Prism 7, and all parameters are expressedas Mean±Standard Error of the Mean (S.E.M.).

Additional Examples

Examples were obtained by carrying out embodiments of the disclosedproducts and methods. A robust microfluidic extrusion setup of FIG. 15and FIG. 16 was designed and used to consistently generate collagenmicrofibers for subsequent testing. This approach yielded continuousmicrofiber production without defects for crosslinking.

To strengthen and stabilize the collagen microfibers, a wide range ofconventional, novel, and combination crosslinking conditions werescreened. Table 2 in FIG. 18 shows a summary of crosslinkers and meanUTS of 50 types of crosslinked microfibers as compared to theun-crosslinked microfibers using the testing method described above.This data showed different crosslinkers/crosslinking protocols(crosslinking in situ or post extrusion, range of crosslinkerconcentrations and time of crosslinking) affected the UTS of themicrofibers to varying degrees. The crosslinking condition that hadsignificantly high mean UTS amongst all the conditions tested with thatcrosslinker has been starred (p<0.01) in Table 2 in FIG. 18.

As shown in FIG. 18, crosslinking procedures with chemicals postextrusion, such as glyoxal (10 mM and 72 hours post extrusion, 121.2±7MPa) and DL-Glyceraldehyde (25 mM and 72 hours post extrusion, 128±12MPa), resulted in microfibers with UTS nearly 20-fold higher than theun-crosslinked microfiber (6.1±1 MPa). Notably, crosslinking using EDCand EDC/NHS on microfluidic microfibers using this extrusion setupyielded UTS values (16.6±2 MPa and 30.2±1 MPa respectively), which issignificantly lower than the glyoxal and DL-Glyceraldehyde groupsdescribed above. In situ crosslinking using chemical crosslinkers suchas choline bitartrate (1 mM or 100 mM), EGCG (200 μM and 1 mM) andD-sorbitol (200 mM) resulted in a significant decrease (p<0.01) in UTScompared to un-crosslinked microfiber. Physical crosslinking techniquessuch as DHT (3 days, 16.2±1 MPa) post extrusion also yielded microfibersstronger than the un-crosslinked microfiber, but was weaker than thechemical crosslinking groups using glyoxal and DL-Glyceraldehydedescribed above. UVR treatment (1.9±0.2 MPa) of methacrylated collagenmicrofibers post extrusion also yielded fibers significantly weaker thanun-crosslinked collagen microfibers (p<0.01).

Since crosslinking of extruded microfibers using glyoxal were amongstthe highest in UTS, further crosslinking of some of the in situ(L-Lysine or D-Sorbitol) or otherwise crosslinked fibers (DHT and UVR)was carried out with 10 mM glyoxal for various time points. Additionalcrosslinking with glyoxal increased the UTS of all these groups withmost significant increase (p<0.01) observed for L-Lysine (10 mM, 2hours)/Glyoxal (10 mM, 24 hours) (96.9±5 MPa) and UVR (0.3hours)/Glyoxal (10 mM, 24 hours) (86.6±10 MPa) groups.

Turning now to FIG. 19, FIG. 20, and FIG. 21, the mechanical propertiesof representative microfibers from crosslinker groups tested in Table 2in FIG. 18 were compared to values reported for human ACL (Noyes andGrood, 1976; Peters et al., 2018), Achilles tendon (Wren et al., 2001)and dermis (Gallagher et al., 2012). FIG. 19 sets forth the UTS, MPA, ingraph 1900. Graph 2000 in FIG. 20 summarizes Modulus I MPa, and graph2100 in FIG. 21 is directed to Strain at Failure, %. The ACL values areshown as line 1930, line 2030, and line 2130. The AT values are shown asline 1910, line 2010, and line 2110, and the dermis values are shown asline 1920, 2020, and 2120. Each result relates to a single microfiber.Results illustrated in FIG. 19 revealed that mean UTS of collagenmicrofibers for some of the crosslinking groups, notably, 10 mM glyoxalwith or without 10 mM L-Lysine in situ and 25 mM DL-Glyceraldehyde areequal to or greater than reported UTS of human ACL, AT, and dermis.

These charts reveal that the mechanical properties of the microfibersextruded as described above can be tuned to match and/or exceed thosefor human Anterior Cruciate Ligament (ACL), human Achilles Tendon (AT),and human dermis by changing crosslinking scenarios. The data isobtained from least 4 identical replicates and error bars indicateS.E.M.

Examples 3 Through 6 Comparative Examples 1, 2 and 3

Four crosslinking conditions shown in italics from the initial screenshown in Table 2 in FIG. 18 and FIG. 19, FIG. 20, and FIG. 21, factoringsalient considerations such as mechanical performance, processing time,and/or cost, for further evaluation. The following fibers areexemplified herein:

Example 3 is telocollagen cross-linked with 10 mM glyoxal for 72 hours(Telo GLY). Example 4 telocollagen cross-linked with 25 mMDL-Glyceraldehyde for 24 hours (Telo DLG). Example 5 is atelocollagencross-linked with 10 mM glyoxal for 24 hours (Atelo GLY). Example 6 isatelocollagen cross-linked with 25 mM DL-Glyceraldehyde for 72 hours(Atelo DLG). Comparative Example 1 is telocollagen cross-linked with0.25 mM EDC for 24 hours (Telo EDC). These groups were compared toun-cross-linked microfibers (Comparative Example 2) and dry Telo GLYfibers (Comparative Example 3). Telo EDC group (Comparative Example 1)was used for comparison as it is a commonly used benign crosslinker inthe field (Cornwell et al., 2007; Enea et al., 2011; Ahmad et al.,2015). Additionally, a high draw collection (high collection speedcompared with raw material feed) onto a grooved solid spool 1110, ascompared with two-bar device 1508, was used to generate thin,ribbon-like microfibers shown in FIG. 22 to further optimize materialproperties.

FIG. 22 shows images of Example 3 Telo GLY microfiber(s) depictingultrastructural features. Frame A is a light microscopy image of asingle dry extruded crosslinked microfiber. Frame B and frame C are SEMimages of a dry single microfiber at different magnifications. Frame Dshows a cross-section of bundled microfibers soaked for 30 minutes inPBS. Frame D reveals structural details and evidence that extrusionusing an embodiment of a novel microfluidic setup disclosed herein andshown in FIG. 15 and FIG. 16, followed by the crosslinking strategy,manufactured consistent, uniform and thin ribbon-like microfibers, asshown by arrows 2206. Arrow 2201 and arrow 2202 in frame B indicatecrevice and ridges along the longitudinal axis of dry microfiber. Arrow2204 and arrow 2205 in frame C show the fibrous sub-fiber structure ofwet microfiber. Frame E, frame F, and frame G represent longitudinal TEMimages of extruded microfibers.

Optimization of crosslinking chemistry and changes in collection methodsled to significant differences in mechanical properties, as summarizedin FIG. 23, FIG. 24, FIG. 25, FIG. 26, FIG. 27, and FIG. 28. Tensiletesting was carried out in the bath and sample holding system describedabove. Width and thickness of DPBS soaked ribbon-like collagenmicrofibers FIG. 23 and FIG. 24 measured from representative images suchas those shown for Example 3 in FIG. 22, frame A, frame B, and frame C,were used calculate the improved UTS, graph 2500 in FIG. 25, and moduluson graph 2600 in FIG. 26. Example 3, Example 4, Example 5, and Example 6were similarly tested, as were Comparative Example 1, ComparativeExample 2, and Comparative Example 3.

When compared to the wet un-crosslinked (34.1±2 μm) ribbon-like collagenmicrofibers, wet Atelo GLY (39.2±1 μm) and Telo EDC (46.4±2 μm)ribbon-like collagen microfibers showed a significantly higher width(p<0.05), as indicated by indicator 2402. Wet Atelo GLY ribbon-likecollagen microfibers were also significantly thicker (11.9±0.5 μm) thanthe un-crosslinked ribbon-like collagen microfibers (9.2±0.5 μm)(p<0.01), as indicated by indicator 2301 in FIG. 23 and indicator 2303in FIG. 23. Thickness of Telo GLY (11.1±0.5 μm), Telo DLG (8.6±0.2 μm)and Atelo DLG (10.9±0.4 μm), and widths of Telo GLY (36.1±0.7 μm), TeloDLG (35.4±0.8 μm) and Atelo DLG (31.1±1 μm) ribbon-like collagenmicrofibers upon soaking in DPBS, were similar to that for theun-cross-linked ribbon-like collagen fiber. The most significant changein UTS shown in graph 2500 of FIG. 25 was observed for un-crosslinkedribbon-like collagen fibers; mean UTS, identified as indicator 2504, andmodulus, identified in graph 2600 of FIG. 26 as indicator 2604,increased from 6.1±1 MPa and 119.8±23 MPa to 35.8±3 MPa and 701±53 MPa.Ribbon-like collagen microfibers from groups such as Telo GLY (121±7 MPaUTS and 1103±63 MPa modulus to 299±15 MPa and 3431±86 MPa respectively)and Atelo DLG (128 MPa UTS and 1734±79 MPa modulus to 231±18 MPa and3408±185 MPa respectively) demonstrated at least a 2-fold increase inmean UTS, as shown in graph 2700 in FIG. 27, and modulus, as shown ingraph 2800 in FIG. 28. There was no change in strain at failure (%) forall groups tested.

There was a significant increase in tensile properties of all theextruded ribbon-like collagen microfibers from a grooved solid spool.Un-cross-linked ribbon-like collagen microfiber group demonstrated thehighest fold change in mean UTS, and modulus compared to othercrosslinker groups. Graph 2700 in FIG. 27 demonstrates the significantfold change of UTS in comparison to the data reported in FIG. 19, FIG.20, and FIG. 21, and graph 2800 in FIG. 28 demonstrates the significantfold change in modulus in comparison to the data reported in Figure FIG.19, FIG. 20, and FIG. 21 for each of Example 3 through Example 6 andComparative Example 1 through Comparative Example 3.

An unpaired two tail t-test was used to assess any significantdifferences between any two groups in FIG. 23, FIG. 24, FIG. 25, FIG.26, FIG. 27, FIG. 28, FIG. 29, FIG. 30, FIG. 31, FIG. 32, FIG. 33, FIG.37, FIG. 38, and FIG. 39. Two-way ANOVA followed by the post-hoc Tukey'sMultiple Comparison Test and unpaired two tail t-test were used toassess differences in UTS for different cross-linker groups in Table 1in FIG. 17. Ordinary one-way ANOVA followed by Dunnett's multiplecomparisons test was performed to assess differences in FIG. 31, FIG.32, and FIG. 33, described in additional detail below. A priori, pvalues <0.05 were defined as significant. All tests were performed usingGraphPad Prism 7, and all parameters are expressed as Mean±StandardError of the Mean (S.E.M.).

Results shown as Mean±S.E.M. and is representative of 3 replicates from2 or more separate experiments. For indicator 2301, p<0.05. Forindicator 2402 and indicator 2502, p<0.01. For indicator 2303, p<0.005.For indicator 2504 and indicator 2604, p<0.0001.

For embodiments of the disclosure, microfiber ultra-structure wasdetermined using light microscope, SEM, and TEM imaging. Other types ofimaging may be used. In Example 3, glyoxal cross-linked telocollagenmicrofibers were characterized. Light microscopy imaging, shown in frameA of FIG. 22, and SEM imaging in frame B of FIG. 22, confirmedhomogenous width of dry microfiber along the longitudinal axis. FIG. 22,frame B, and high magnification SEM (frame C of FIG. 22) imaging oflongitudinal section revealed parallel alignment of ridges and creviceswithin the dry microfiber, as shown by FIG. 22, frame D. Frame D of FIG.22 highlights cross-sectional features of DPBS soaked extrudedcrosslinked microfiber bundle using SEM. These images revealultrastructural features of an external smooth surface with apparentfibrous sub-fiber structure, as shown at arrows 2206. This demonstratesthat extruded crosslinked microfibers are consistent, thin, andribbon-like. Further evidence that collagen alignment from themolecular-through nano-scale in native connective tissue isrecapitulated in our crosslinked microfibers is revealed from TEMimaging, frame E, frame F, and frame G of FIG. 22.

To biochemically assess the degree of crosslinking, biochemical, andbiophysical characterization of embodiments of crosslinked microfibers,a ninhydrin assay was used. The results are set forth in graph 2901 inFIG. 29. Example 3, Telo GLY (86±1%) and Example 6, Atelo DLG (82±3%)microfibers demonstrated significantly higher degree of crosslinkingcompared to Example 5, Atelo GLY (68±4%) and Example 4, Telo DLG(59±6%), highlighting that higher time of crosslinking improvedcrosslinking efficiency.

Primary and secondary protein structure of the extruded collagenmicrofibers also was assessed. SDS-PAGE analysis of the acidifiedstarting material confirmed the presence of primary alpha, beta andgamma chains of collagen. However, due to the inability of themicrofibers to be dissolved in acid, it was not possible to detect anycollagen in the microfiber acid extracts.

Biophysical characterization using differential scanning calorimetry(DSC) measurements on extruded microfibers revealed an insignificantincrease in melting temperatures between the un-crosslinked and thecrosslinked microfiber groups, as depicted in graph 2902 in FIG. 29 forthe same samples tested for graph 2901. However, the average meltingtemperature of all the extruded microfibers (74±3° C.) (line 2930 ongraph 2902) was significantly higher than that for the human AT (line2920 on graph 2902) (60° C.) (Wiegnad, Patczai and Lorinczy, 2017),indicative of greater overall structural stability (Sanchez-Ruiz, 1995).FTIR spectra in graph 2903 of FIG. 29 showed no significant peak shiftsin the amide I (1650 cm⁻¹), amide II (˜1560 cm⁻¹), amide III (˜1235cm⁻¹), amide A (˜3285 cm⁻¹) and amide B (2917 cm⁻¹) regions, indicatingthat the secondary structure of microfibers was unchanged after theextrusion as well as the crosslinking process used in this disclosure.Data in graph 2901 is shown as Mean±S.E.M. and is representative of 3replicates from 2 separate experiments. In graph 2901, indicator 2910depicts p<0.05.

Cellular attachment, metabolic activity, and cytotoxicity of theextruded microfibers also were determined for embodiments of thedisclosure, as shown in illustrations 3001 and 3002 of depiction 3000 inFIG. 30, and in FIG. 31, FIG. 32, and FIG. 33. These figures includeExample 3 through Example 6, Comparative Example 2, and other samples.Human tenocytes were used to assess collagen fiber cytocompatibility, asdescribed above. The attachment of tenocytes on Telo GLY microfiberswith elongated morphology (Example 5) is shown in illustration 3001 inFIG. 30. About 70% of tenocytes seeded on the Telo GLY microfibersremained attached after 12 days. No significant change in tenocytesmetabolic activity over 7 days was noted by AlamarBlue fluorescence, assummarized in FIG. 31 for Example 3 through Example 6, ComparativeExample 1, and other samples, when compared to the positive control(Cells only group). However, metabolic activity for cells growing withmicrofibers from selected fiber groups was significantly higher (p<0.05)than that for negative controls (10 mM glyoxal chemical and ZDBC film).As illustrated in FIG. 32, viability of tenocytes incubated withmicrofibers was between 75% to 85% compared to tenocytes growing onplastic (100%) when assayed using the MTT reagent. The negative controls(10 mM GLY chemical and ZDBC film) demonstrated significantly lower(p<0.005) tenocyte survival than the “Cells Only”, Telo DLG (Example 4),Atelo GLY (Example 5), and Telo GLY (Example 3) groups. Similar resultswere observed using LDH assay (FIG. 33), wherein the all the extrudedmicrofiber groups except for Atelo DLG and Telo DLG elicited tenocyteviability similar to the “Cells Only” group. As indicated at indicator3303 of FIG. 33, at the end of 7 days, the 10 mM GLY chemical group didnot have enough tenocytes (ND) to be assayed by LDH release into themedia. A commercially available coated Vicryl suture from Ethicon,typically recommended in wound closure, provided a comparison. Resultsindicated that microfiber embodiments of the disclosure exhibitedsignificantly lower cytotoxicity (p<0.005) than the suture using bothLDH and MTT assays (FIG. 32 and FIG. 32). Overall, multiple assays wereused to establish cytocompatibility of the extruded microfibers.

In particular, image 3001 and image 3002 show representative confocalimages of human tenocytes attached to Telo GLY microfibers (Example 3),with DAPI (arrows 3005) and live cell stain (CMFDA, as shown at arrows3003), respectively, showing cytoplasmic extensions and elongatednuclei. FIG. 31 shows no significant change in metabolic activity ofhuman tenocytes incubated with the crosslinked microfibers assayed usingAlamarBlue after 7 days of incubation compared to the cells only group.Metabolic activity was significantly lower in tenocytes incubated withnegative controls (ZDBC film and 10 mM GLY chemical) and Vicryl suturein comparison to the microfiber groups. MTT assay results summarized inFIG. 32 revealed a decrease in viability for tenocytes incubated withthe microfiber groups compared to the cells only group but a significantincrease compared to negative controls. On the other hand, LDH assayresults shown in FIG. 33 show a significant decrease in cell survivalfor the Atelo DLG (Example 6) and Telo DLG (Example 4) microfiber groupsas well as the negative controls. Both MTT and LDH assays were performedat 7 days post incubation with tenocytes. All data in FIG. 32 and FIG.33 was normalized to the cells only group. (ND) at indicator 3303indicates that the 10 mM Glyoxal chemical treatment group had asignificant arrest in proliferation resulting in insufficient number ofcells to detect LDH at the end of the assay timepoint. In these figures,indicator 3101 and indicator 3301 show p<0.05, indicator 3202 showsp<0.01, indicator 3103 and indicator 3203 identifies p<0.005, andindicator 3204 and indicator 3304 identify p<0.0001).

To assess biocompatibility of extruded microfiber embodiments of thedisclosure, sterilized microfiber bundles of the selected 4 cross linkergroups of Example 3 through Example 6 (Atelo DLG, Telo DLG, Telo GLY andAtelo GLY) were subcutaneously implanted in rats per ISO 10993-6.Microfiber bundles implanted from each of the 4 crosslinker groups inFIG. 23, FIG. 24, FIG. 25, FIG. 26, FIG. 27, and FIG. 28, and the suturecontrol (collagen coated FiberWire®) group elicited a distinct hosttissue response characterized by different extents of cellularinfiltration, vascularization, collagen deposition and tissue remodelingshown in FIG. 34, FIG. 35, and FIG. 36. Amongst these, glyoxalcross-linked microfiber groups (Telo (GLY), Example 3, or Atelo (GLY)Example 5) showed lower pro-inflammatory response in comparison toDL-Glyceraldehyde (Telo DLG, Example 4, or Atelo DLG, Example 6)cross-linked groups. Representative H & E staining images of the TeloGLY (Example 3) group shown in transverse image 3401 and longitudinalimage 3402 of FIG. 34 demonstrated significantly high cellularinfiltration compared to the suture control shown in FIG. 36, includingtransverse image 3601 and longitudinal image 3602. The suture control inFIG. 36 elicited an intense inflammatory response at 4 weeks compared tothe microfiber implants.

Deposition of newly formed collagen was visualized by Masson's Trichromestaining in native tissue surrounding the Telo (GLY) (Example 3)microfiber implants in image 3501 of FIG. 35. Longitudinal sectionstained with Masson's Trichrome is as shown in image 3502 of FIG. 35, aswell as polarized light imaging of image 3602 of FIG. 36, showdeposition of newly formed collagen around the microfibers in anorganized fashion.

Blood vessels and capillaries were identified within the microfiberimplants and in surrounding tissues as observed in higher magnificationcross-sectional images of H & E stained sections (arrows in image 3401of FIG. 34).

FIG. 34, FIG. 35, and FIG. 36 are representative images of ratsubcutaneous implants at 4 weeks for the Telo GLY (Example 3) group.Image 3401 and image 3501 show microfibers m, identified by arrows 3410,on stained slides illustrating transverse sections of H&E in FIG. 34 andMasson's Trichrome in FIG. 35. Inset 3490 shows the entire section ofthe H&E implant, and inset 3495 shows the portion depicted in image3401. Similarly, inset 3590 shows the entire section of the Masson'sTrichrome implant, and inset 3595 shows the portion depicted in image3501. Both image 3401 and image 3501 illustrate significant cellularinfiltration. Arrows 3420 in FIG. 34 point at blood vessels within theimplants. Image 3601 shows negligible cellular infiltration in controlsamples, collagen coated FiberWire® using H&E staining. Image 3402 showsH&E and image 35032 shows Masson's Trichrome staining for longitudinalsections of the microfibers' implant. Polarized light image 3602 in FIG.36 shows aligned microfibers as well as newly formed collagensurrounding the implants. Image 3402, image 3502, and image 3602demonstrate the formation of new aligned collagen in native tissuesurrounding the implants. The legend “Suture Control” on image 3601identifies the collagen coated FiberWire®.

Immunostaining was used to determine extents of macrophage polarizationin native tissue around microfiber implants from 4 crosslinker groups,FIG. 37 and FIG. 38 are representative immunofluorescent images showingexpression patterns of CCR7 (M1) (image 3700 on FIG. 37) and CD163 (M2)(image 3800 on FIG. 38) macrophage phenotype in the native tissue ofrats surrounding Telo GLY (Example 3) microfiber implants at 4 weeks.FIG. 38 shows quantitation of the percentage of macrophages thatexpressed both M1 and M2, M1 only, M2 only, or no M1/M2 phenotype.Glyoxal cross-linked groups (Telo (GLY), Example 3, and Atelo (GLY),Example 5) demonstrated significantly higher proportion of macrophagesexpressing M1 and M2 phenotype (about 40%) compared to theDL-Glyceraldehyde cross-linked groups (Telo DLG, Example 4, and AteloDLG, Example 6), as shown in graph 3900 of FIG. 39. Furthermore, betweenthe Telo (GLY), Example 3, and Atelo (GLY), Example 5, groups, Telo GLYimplants elicited a small subset of cells expressing M2 only phenotype(6%), while the rest of the groups had negligible M2 only phenotype;Atelo GLY (0.2%), Telo DLG (0%) and Atelo DLG (0%) (FIG. 39). There wasa significantly higher proportion of cells with M1 phenotype in theDL-Glyceraldehyde crosslinked groups; Telo DLG (64%) and Atelo DLG (58%)compared to the glyoxal crosslinked groups; Telo GLY (24%) and Atelo GLY(19%). Staining with appropriate controls, as described above, revealednegligible non-specific background staining (not shown). Sectioningartifacts of the suture control samples, and significant backgroundstaining, made it difficult to perform this analysis on these samples.

Representative immunofluorescent image 3700 and image 3800 show examplesof the host macrophage response to the Telo (GLY), Example 3,microfibers, identified as m at arrows 3840, at 4 weeks. Arrows 3710 andarrows 3810 indicate examples of cells expressing both M1 and M2. Arrow3720 and arrow 3820 indicate examples of cells expressing M1 only.Arrows 3730 and arrows 3830 indicate cells expressing M2 only phenotype.Arrows 3840 point to microfiber bundles, identified by m. Graph 3900 ofFIG. 39 shows the % of cells expressing M1 and M2, M1 only, M2 only, orno M1/M2 phenotype for the 4 groups of cross-linked microfibers. Resultsfrom this analysis show initiation of pro-regenerative M2 macrophagephenotype in all microfiber groups tested. Glyoxal cross-linked fibergroups showed higher proportion of cells with M1 and M2 phenotypecompared to the DL-Glyceraldehyde cross-linked fiber groups.Furthermore, Telo GLY group had a small but significant subset of M2only macrophages. In graph 3900, indicator 3901 identifies p<0.05,indicator 3902 indicates p<0.01, and indicator 3903 indicates p<0.005).

The effect of long-term hydration was determined for embodiments of thedisclosure of microfibers in culture media on mechanical properties anddegree of swelling. Because Telo (GLY), Example 3, microfibers showedoptimal mechanical properties, cytocompatibility, and biocompatibility,this group was further tested for long-term stability mimicking in vitrophysiological conditions. Incubation in EMEM (Eagle's Minimum EssentialMedium) led to increase in microfiber width by 53% (36.4±1.1 μm, Day 0to 56.0±1.6 μm, 6 months) in 6 months as shown by graph 4000 in FIG. 40.This graph shows the degree of swelling of wet microfibers over time.The swelling was accompanied by a significant loss in mechanicalproperties. Graph 4100 in FIG. 41 shows that mean force at failuredecreased by 54% from its initial value in 6 months. Mean UTS (graph4200 in FIG. 42) and modulus (graph 4300 in FIG. 43) also reduced by 82%from the starting timepoint in 6 months. There was no significant changein strain at break (%) between Day 0 and 6 months of incubation (graph4400 in FIG. 44).

Therefore, FIG. 40, FIG. 41, FIG. 42, FIG. 43, and FIG. 44 show thatTelo GLY microfibers were stable and did not dissolve when incubated inconditions mimicking in vitro biological environment (sterile cellculture media in a humidified incubator maintained at 37° C. and 5% CO₂)for up to 6 months.

As can be seen from the figures, mechanical stability of Telo GLYmicrofibers incubated in sterile EMEM and under tension assessed after 1week, 1 month, 3 months and 6 months in a humidified incubator at 37° C.and 5% CO₂ shows that Telo GLY microfibers at the end of 6 months swellby 50% (FIG. 40), lost 60% of the Force at Failure (FIG. 41), lost 80%of UTS (FIG. 42), and lost 80% of Modulus (FIG. 43) compared to Day 0.However, there was no significant change in % Strain at failure at theend of 6 months (FIG. 44). All the values depicted in these figures havebeen normalized to a value of 1 for Day 0. The continuous lines in thesefigures have been drawn by inspection only to serve as a guide to thereader. Data shown as mean±S.E.M. and is representative of at least 5replicates.

SDS-PAGE was used to compare the collagen starting materials(lyophilized telo- or atelo-collagen), un-cross-linked, and cross-linkedmicrofibers. Collagen starting materials readily dissolved in 50 mM HClafter overnight agitation. However, the extruded microfibers did not gointo solution at a concentration of 0.5 mg/ml and so provided no bands.To confirm the presence or absence of collagen in acid extracts ofmicrofibers, these extracts were run with the solution of startingmaterial and a pre-stained molecular weight marker (HiMark, Invitrogen,CA) on a gradient gel (3%-8%) (Invitrogen). SimplyBlue™ (Invitrogen, CA)was used to stain gels followed by rinses with deionized water tode-stain them. The gels were then imaged under white light to view allvisible protein bands. Thus, SDS-PAGE shows that the extruded fiber fromgroups with maximum UTS and un-cross-linked fibers were resistant toacid hydrolysis when compared to the acidified starting materials thatshow type I collagen fingerprint with bands characteristic of monomericregions at about 115 kDa, dimeric regions at about 230 kDa, and trimericregions at about 460 kDa regions.

FIG. 45 summarizes mechanical tensile properties of some of the bestperforming (hydrated) cross-linked collagen fibers published inliterature, compared to an embodiment of the disclosure.

In summary, this disclosure is directed to a novel microfluidicextrusion process to manufacture type I collagen microfibers withprecision, consistency, and scalability as biocompatible fibers to beused in indications ranging from natural sutures to engineeredconnective tissue. This disclosure reveals the biomanufactured glyoxalcrosslinked telocollagen microfiber embodiments of the disclosuredemonstrate dry and wet-tensile properties superior to prior crosslinkedcollagen extruded microfibers (Paul and Bailey, 2003; Caruso and Dunn,2004; Zeugolis, Paul and Attenburrow, 2009; Enea et al., 2011).

While many prior studies do not report whether tensile testing wasperformed on hydrated fibers or provide arguably misleading results fordry fibers, or does not disclose how the fibers were wetted if fullyhydrated, results of embodiments of the disclosure herein provide dryand hydrated properties of optimized crosslinked fibers with a detailedmethodology for testing, which is critical for comparisons and growthwithin the field.

Collection of fibers on a grooved drum led to significant alterations instructural and hydrated mechanical properties of all the crosslinkedmicrofiber (see FIG. 22, FIG. 23, FIG. 24, FIG. 25, FIG. 26, FIG. 27,and FIG. 28). Mechanically, this improved strength may be related totempering, thinning, and improved molecular alignment to the ribbonswhich were once fibers, resulting in fiber tensile properties strongerthan the ACL, Achilles tendon, dermis, or any other soft connectivetissue.

In embodiments of the disclosure, determining the degree of crosslinkingmechanism efficiency is emphasized. Insufficient crosslinking can leadto lower tensile strengths while overuse of chemical crosslinker canlead to residues of crosslinker on the surface of the microfibersresulting in cytotoxicity. The ninhydrin assay (FIG. 36) revealed thatgroups with maximum crosslinking degree were those that were crosslinkedfor 72 hours (Telo GLY and Atelo DLG), which also correlated with asignificant increase in tensile strength. The chemistry of crosslinkingusing aldehydes involves the formation of Schiff's base type compoundswith functional amino groups in collagen, leading to strong molecularbonds (Fathima et al., 2004).

Chemical analysis of embodiments of extruded microfibers revealed thatthese were further resistant to acid hydrolysis. The microfluidicsapparatus, or setup, disclosed herein generated microfibers withchemical stability higher than the lyophilized starting materialsuggesting tight packing of the collagen molecules in the microfibersresulting in a stable higher order structure and suggests low internalmoisture content. Such higher order structure has been reported innative connective tissues (Benjamin, Kaiser and Milz, 2008; Wang, Guoand Li, 2012). Integrity of the secondary structure in the extrudedmicrofibers was confirmed from FTIR analysis shown in FIG. 29 which alsosuggested that neither the extrusion process nor the crosslinkingtechnique denatured the collagen.

While crosslinking of collagen in a biomimetic might help improvingtensile properties, degradation of chemicals (e.g. glutaraldehyde) usedfor crosslinking can be toxic (Gough, Scotchford and Downes, 2002;Umashankar, Kumari and Mohanan, 2012). Amongst other chemicals thatexhibit somewhat less cytotoxicity, EDC or EDC/NHS as crosslinker hasbeen a popular basic research choice for collagen microfibers (Enea etal., 2011; Ahmad et al., 2015; Shepherd et al., 2015). However, there isonly a negligible improvement in tensile strength and noted toxicitywith these classic crosslinkers, making them poorly suited for use inconnective tissue repair. We show in this study the development ofmechanically superior extruded collagen microfibers chemicallycrosslinked with either glyoxal or glyceraldehyde, which are highlycytocompatible (see FIG. 30, FIG. 31, FIG. 32, and FIG. 33) andbiocompatible in vivo as shown in FIG. 34, FIG. 35, and FIG. 36), perstandardized ISO 10993 testing as is commonly required for USFDAapproval. Furthermore, collagen microfibers cross-linked with glyoxalare resistant to acid hydrolysis, demonstrate ultrastructural featuresdown to the near molecular level, and remain stable in cell culturemedia for at least 6 months with the ability of a single microfiber toretain around between about 30% and about 50%, typically about 40%, oftheir initial load carrying capacity and a UTS value higher than nativeACL (see FIG. 40, FIG. 41, FIG. 42, FIG. 43, and FIG. 44). Augmentingsuture repair of ACL or Achilles tendon with collagen based microfibersor using a collagen-based braided suture in wound healing as describedherein requires the collagen-based material not only to support thetissue mechanically but also to promote tissue remodeling at areasonable rate (Dunn, Avasarala and Zawadsky, 1993). Therefore, forbiomedical application, in vitro and/or in vivo biocompatibility testsare critical to establish the effects of these chemically crosslinkedmicrofibers on cytotoxicity, inflammatory response, and regenerativeresponse. Embodiments of extruded microfiber bundles of the disclosurewere cytocompatible and demonstrate minimal toxicity to human tenocytes.Microfluidic extruded microfibers of the disclosure further supportedthe attachment of human tenocytes and assumed the elongated shape asobserved on connective tissue (Benjamin, 2010). Biocompatibility hasbeen defined as the ability of an implant to “locally trigger and guidenon-fibrotic wound-healing, reconstruction and tissue integration”(Ratner, 2011). Cross-linked microfiber bundle implants followingsubcutaneous implantation in rats for 4 weeks manifested low (glyoxalgroups) to moderate (glyceraldehyde groups) inflammatory response, withthe glyoxal-telocollagen group demonstrating initiation of apro-regenerative response. Additionally, long-term stability data andrat histology images indicated stability of the microfibers for up to atleast 6 months in vitro and 4 weeks in vivo. Thus, embodiments of thedisclosure are able to maintain strength in vivo for at least about 1month and in vitro for at least about 3 months, and up to about 6 months

Macrophages are a heterogenous mix of mononuclear cells that areactivated in the host as a response to tissue damage (Mosser, 2003;Gordon and Taylor, 2005) such as, during implantation of materials.Macrophage phenotype polarization at the interface of the implant andthe host tissue (Kasner et al., 2009; Brown et al., 2012) is importantin determining the potential of the host to overcome pro-inflammatorysignals and transition towards tissue repair and remodeling in responseto the surgical implant. Macrophage phenotype has been broadlycharacterized as M1, or “classically” activated, possessingpro-inflammatory signals and M2, or “alternatively” activated,possessing immunoregulatory or tissue remodeling characteristics (Millset al., 2000). However, it is important to note that activatedmacrophages possess plasticity in a way that they are able to switchfrom M1 to M2 and from M2 to M1 phenotypes easily. This plasticity istriggered by changes in the local microenvironment (Porcheray et al.,2005; Stout et al., 2005). Due to this, macrophages may also adopttransitional characteristics of both M1 and M2 phenotype (Brown andBadylak, 2013). In embodiments of the disclosure, the proportion ofcells exhibiting M1, M1 and M2, or M2 phenotypes were determined. Thesedeterminations suggest the following: (1) at 4 weeks of implantation theglyoxal crosslinking groups had cells with more M1 and M2 or M2 onlyphenotype indicating that a tissue remodeling response had beeninitiated by the host at 4 weeks. This suggests that the microfibersfrom the glyoxal groups were most biocompatible. To the best of ourknowledge, such in depth analysis of immunologic response has not beenperformed using crosslinked collagen microfibers.

Incorporation of collagen into sutures for wound healing has been achallenge. However, this disclosure provides a method and apparatus formanufacture of effective products. A collagen coated FiberWire®non-resorbable suture (the only collagen based synthetic sutureavailable in the market) was used as control for investigation herein.This FiberWire® showed limited cellular infiltration leading to verylittle ingrowth or regeneration of native tissue around the implants. Incontrast, glyoxal cross-linked collagen microfiber embodiments of thedisclosure in the form of suture-like bundles showed significantcellular infiltration with newly-formed collagen in the surroundingtissue, indicative of regenerative healing.

Embodiments of the disclosure illustrate that microfluidic extrusion oftype I clinical quality collagen fibers cross-linked with glyoxalexhibit exemplary tensile strength, structural stability,cytocompatibility, and biocompatibility, exceeding prior reported purecollagen made by other biomanufacturing processes. Using glyoxal tostabilize collagen fibers presents a clinically relevant, safe, andeffective method for additive biomanufacturing of collagen microfibers.These optimized collagen microfibers can readily be manufactured intodiverse biomedical applications ranging surgical suture, ligamentinternal braces, tissue engineered ligaments, tendons and other strong,fibrous tissues, designed for significantly improving human health.

Example 7

Collagen solution and formation buffer were prepared. Clinical gradelyophilized atelocollagen (Symatese, France) in an amount sufficient toform a solution having a concentration of 1.6% (w/v) was dissolved in0.05 M acetic acid in a closed polypropylene container. The solution wasstirred overnight at room temperature at 180 rpm. The total volume ofthe solution was less than half of the volume of the container to ensureuniform mixing. On the next day, the acidified collagen mixture was spundown in a centrifuge at 730 g for 5 minutes. The solution was degassedfor 2 minutes and spun down for 10 minutes at 730 g to remove bubbles.The resultant acidified atelocollagen was pulled up into eight 20 mLsyringes (Hsw® Norm-Ject® Sterile Luer-Lock Syringes, VWR) to be useddirectly with high-output collagen microfiber extrusion equipmentillustrated in FIG. 46.

To prepare the formation buffer, to 100 ml of Milli-Q water, 10 gm PEG(polyethylene glycol) (35 KDa, ChemCruz), 0.686 gm TES(N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid) (SigmaAldrich), 0.790 gm Sodium Chloride (Sigma Aldrich), 0.414 gm SodiumPhosphate Monobasic (Baker Analyzed), and 1.21 gm Sodium PhosphateDibasic (Sigma Aldrich) were added. This mixture was stirred overnightat room temperature in a glass beaker on a stir-plate at 400 rpm. On thenext day, the pH of this solution was adjusted to 8 by adding 10 MSodium Hydroxide (Sigma Aldrich) and the solution then was filteredusing a 0.45 μm filter.

On the day of the extrusion, to 800 ml of Milli-Q water, 200 ml ofethanol (Fisher Scientific) was mixed to obtain a 20% ethanol solutionfor the dehydration bath.

FIG. 46 shows a portion of system 4600, in which the acidifiedatelocollagen was processed. A syringe array pump was mounted tocooperate with rotatable plate 4601 and all 8 syringes. Example fiberbundles were made with and without twisting. The fiber bundle travelledthrough the formation bath and became stronger as the buffering solutionneutralized the acid and formed fibrils. The twisted and non-twistedbundles then entered the 20% aqueous ethanol dehydration bath, whichremoves water and further strengthens the fiber. A tensioning rig keepsconstant tension on the fiber bundle until the bundle is deposited ontoa grooved spool (not shown) at the end of the bath. The tension in thefibers aided in drawing and organizing the collagen for strength andstability. The spooled collagen then was dried, crosslinked in glyoxal,and used for forming 3D grafts.

For post-extrusion chemical crosslinking, un-crosslinked taut collagenfiber bundles were collected on big grooved spools were air dried forhalf hour and then submerged into a solution of crosslinker in 70%ethanol solution in a large acrylic tube and then placed on a rocker at1 rpm. The aqueous ethanol medium ensured that microfibers remaineddehydrated throughout the crosslinking period. After crosslinking,microfibers were stored in a desiccator until further tests wereperformed.

The chemical crosslinker used was glyoxal, a dialdehyde, at aconcentration of 10 mM. The chemistry of crosslinking using aldehydesinvolves the formation of Schiff's base type compounds with functionalamino groups in collagen, leading to strong molecular bonds.

Mechanical properties of the thus-produced single fiber bundles weregenerated using a “discrete fiber” test method wherein thecross-sectional area of individual fiber bundles and a known quantity offiber bundles on a cartridge were averaged to determine the ultimatetensile strength (UTS), modulus, and strain at failure (%). Diameters offibers were measured from analyzing images obtained at 10 differentpoints on 3 separate, 1.5-inch long, fiber bundles using an invertedlight microscope (Axio Vert.A1 Model, Zeiss, Germany) and ImageJsoftware (NIH Shareware, Bethesda, Md.).

FIG. 47 through FIG. 51 show results from mechanical testing of thefiber bundles under different test conditions, i.e., for non-twistedfibers and for twisted fibers. There were no significant differencesbetween fiber types. FIG. 52 through FIG. 56 show results frommechanical testing of non-twisted fiber bundles after cross-linking inglyoxal at various times. There were differences at levels p<0.01 (**)in peak load (FIG. 53) and UTS (FIG. 55) and at p<0.001 (****) inmodulus (FIG. 54) and UTS (FIG. 55).

FIG. 57 is a cross-sectional image of microfiber bundles obtained usinga Scanning Electron Microscope (SEM). FIG. 57 illustrates the structureof two multi-fiber bundles. First bundle 5701 and second fiber bundle5702 comprise 8 fibers. First bundle 5701 clearly illustrates firstfiber 5710, second fiber 5720, and third fiber 5730. FIG. 57 alsodistinctly shows the second end 5721 of second fiber 5720, third end5721 of third fiber 5730, and fourth end 5739 of a fiber that isotherwise not distinctly identifiable. Surface 5705 is the ends of allof the fibers in the first fiber bundle.

Second fiber bundle 5702 shows fifth fiber 5740 and fifth end 5741;sixth fiber 5750 and sixth end 5751; seventh fiber 5760 and seventh end5761; and eighth end 5749 of a fiber that is otherwise not distinctlyidentifiable. Surface 5706 is the end of all of the fibers in secondfiber bundle 5702.

FIG. 58 is an SEM image of an octa-fiber bundle.

While various embodiments of the invention have been described, thedescription is intended to be exemplary, rather than limiting and itwill be apparent to those of ordinary skill in the art that many moreembodiments and implementations are possible that are within the scopeof the invention. Accordingly, the invention is not to be restrictedexcept in light of the attached claims and their equivalents. Also,various modifications and changes may be made within the scope of theattached claims.

REFERENCES

All documents identified in this specification, including the followingarticles and patent properties, are incorporated by reference in theirentireties.

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We claim:
 1. An extruded collagen fiber comprising: a plurality ofextruded sub-fibers; wherein the sub-fibers are cross-linked withglyoxal.
 2. The extruded collagen fiber of claim 1, wherein the fiberexhibits an ordered, longitudinally oriented structure.
 3. The extrudedcollagen fiber of claim 2, wherein the extruded collagen fiber has oneor more of the following characteristics: an ultimate tensile strengthof between about 1 MPa to about 1,700 MPa; a modulus of elasticity ofbetween about 10 MPa to about 20,000 MPa; a strain at break of betweenabout 2 percent and about 45 percent elongation; an average fiberdiameter between about 10 μm and about 90 μm; maintains its strengthafter soaking in Dulbecco's Phosphate Buffered Solution (“DPBS”) at roomtemperature for at least about 1 hour; retains fifty percent of itsinitial load-bearing capacity for three months in culture; and promotesan elevated pro-regenerative M2 macrophage response in vivo.
 4. Theextruded collagen fiber of claim 3, wherein the fiber is telocollagenhaving a degree of cross linking of at least about 85 percent oratelocollagen having a degree of cross linking of at least about 65percent.
 5. The extruded collagen fiber of claim 3, wherein the ultimatetensile strength is between about 25 MPa to about 1,700 MPa; the modulusof elasticity is between about 15,000 MPa to about 29,000 MPa; and astrain at break of between about 7 percent and about 20 percentelongation.
 6. The extruded collagen fiber of claim 2, wherein thecollagen is selected from the group consisting of clinical gradecollagen, atelocollagen, telocollagen, recombinant collagen, and blendsthereof.
 7. The extruded collagen fiber of claim 2, wherein the collagenfurther comprises one or more bio-acceptable polymers.
 8. The extrudedcollagen fiber of claim 2, wherein the fiber maintains a strengthgreater than about 60 MPa after 6 months in DBPS at room temperature. 9.The extruded collagen fiber of claim 2, further comprising adheredtenocytes; wherein the tenocytes retain at least about 75% cellviability and at least about 95% cell survival after about seven daysincubation under conventional mammalian cell culture conditions oftemperature, pH, and humidity.
 10. The extruded collagen fiber of claim2, wherein the fiber has a cross section that is substantially circular,ovoid, square, rectangular, ribbon-like, triangular, or irregularlyshaped.
 11. A plurality of extruded collagen fibers of claim 2, whereinthe fibers are associated in a form factor that is bundled, braided, orinterwoven.
 12. The plurality of fibers of claim 11, wherein the formfactor is a bundle.
 13. The plurality of fibers of claim 12, wherein thecollagen fibers in the bundle are twisted.
 14. The plurality of fibersof claim 12, wherein the collagen fibers in the bundle are non-twisted.15. The plurality of fibers of claim 11, wherein the form factor is abraid.
 16. The plurality of fibers of claim 11, wherein the form factoris a weave.
 17. The plurality of fibers of claim 11, wherein thecollagen is selected from the group consisting of clinical gradecollagen, atelocollagen, telocollagen, recombinant collagen, and blendsthereof.
 18. The plurality of fibers of claim 11, further comprisingadhered tenocytes; wherein the tenocytes retain at least about 75% cellviability and at least about 95% cell survival after about seven daysincubation under conventional mammalian cell culture conditions oftemperature, pH, and humidity.
 19. The plurality of fibers of claim 11,wherein the fibers maintain a strength greater than about 60 MPa after 6months in DPBS at room temperature.
 20. The plurality of fibers of claim11, wherein the fibers maintain a strength greater than about 60 MPaafter 6 months after implantation into a subject.
 21. The plurality offibers of claim 11, wherein the collagen further comprises one or morebio-acceptable polymers.
 22. A plurality of extruded collagen fibers,the fibers being associated in the form factor of a bundle; whereinsub-fibers of the fibers are cross-linked with glyoxal; wherein thefibers exhibit an ordered, longitudinally oriented structure; andwherein the extruded collagen fiber has one or more of the followingcharacteristics: ultimate tensile strength is between about 1 MPa toabout 800 MPa; the modulus of elasticity is between about 10 MPa toabout 7,500 MPa; and the average fiber diameter is between about 10 μmand about 30 μm.
 23. The plurality of fibers of claim 22, wherein thefibers in the bundle are twisted.
 24. The plurality of fibers of claim22, wherein the fibers are non-twisted.
 25. The plurality of fibers ofclaim 22, wherein the collagen is selected from the group consisting ofclinical grade collagen, atelocollagen, telocollagen, recombinantcollagen, and blends thereof.
 26. The plurality of fibers of claim 22,further comprising adhered tenocytes; wherein the tenocytes retain atleast about 75% cell viability and at least about 95% cell survivalafter about seven days incubation under conventional mammalian cellculture conditions of temperature, pH, and humidity.
 27. The pluralityof fibers of claim 25, comprising between 2 fibers and about 10,000fibers.
 28. The plurality of fibers of claim of claim 22, wherein thefiber maintains a strength greater than about 60 MPa after 6 months inDBPS at room temperature.
 29. The plurality of fibers of claim of claim22, wherein the fiber maintains a strength greater than about 60 MPaafter implantation into a subject.
 30. The plurality of fibers of claim22, wherein the collagen further comprises one or more bio-acceptablepolymers.